Lincomycin derivatives possessing antibacterial activity

ABSTRACT

Novel lincomycin derivatives are disclosed. These lincomycin derivatives exhibit antibacterial activity. As the compounds of the subject invention exhibit potent activities against bacteria, including gram positive organisms, they are useful antimicrobial agents. Methods of synthesis and of use of the compounds are also disclosed.

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/403,770 filed on Aug. 15, 2002, thedisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to lincomycin derivatives that exhibitantibacterial activity.

2. State of the Art

Lincomycin is a biosynthetic product that adversely affects growth ofvarious microorganisms, in particular gram positive bacteria. Thecharacteristics and preparation of lincomycin are disclosed in U.S. Pat.No. 3,086,912. A variety of derivatives of lincomycin, which also haveantimicrobial activity, have been prepared. These derivatives include,for example, clindamycin, which is described in U.S. Pat. No. 3,496,163.

Lincomycin derivatives remain attractive targets for antibacterial drugdiscovery. Accordingly, lincomycin derivatives that possessantimicrobial activity are desired as potential antibacterial agents.

SUMMARY OF THE INVENTION

The present invention provides lincomycin derivatives that possessantibacterial activity.

In one of its composition aspects, this invention is directed to acompound of formula (I):

wherein:

R¹ is alkyl;

R² and R³ are independently H, alkyl, hydroxy, fluoro, or cyanoalkyl orone of R² and R³ is ═NOR⁷ and the other is absent, or one of R² and R³is ═CH₂ and the other is absent, with the provisos that both R² and R³are not H; when one of R² and R³ is fluoro, the other is not hydrogen orhydroxy; and when one of R² and R³ is hydroxy, the other is not fluoro,hydrogen, or hydroxy;

R⁶ is selected from the group consisting of H, alkyl, hydroxyalkyl,—C(O)O-alkylene-cycloalkyl, —C(O)O-alkylene-substituted cycloalkyl,—C(O)O-alkyl, —C(O)O-substituted alkyl, —C(O)O-aryl, —C(O)O-substitutedaryl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl,—[C(O)O]_(p)-alkylene-heterocycle, —[C(O)O]_(p)-alkylene-substitutedheterocycle, wherein p is 0 or 1 with the proviso that—C(O)O-substituted alkyl does not include the following:

R⁷ is H or alkyl;

R⁹, which can be singly or multiply substituted in the ring on the sameor different carbons, is independently selected from the groupconsisting of alkyl, substituted alkyl, alkoxyalkoxy, cycloalkyl,substituted cycloalkyl, alkoxyalkoxy, substituted oxygen, substitutednitrogen, halogen, phenyl, substituted phenyl, —(CH₂)_(n)—OH,—(CH₂)_(n)—NR⁴R⁵, -alkylene-R^(a) where R^(a) is selected frommonofluorophenyl and monochlorophenyl, and branched chain isomersthereof wherein n is an integer of from 1 to 8 inclusive and R⁴ and R⁵are H or alkyl; and

m is 1 or 2; and

prodrugs, tautomers or pharmaceutically acceptable salts thereof;

with the proviso that the compound of formula I has a minimum inhibitionconcentration of 32 μg/mL or less against at least one of the organismsselected from the group consisting of Streptococcus pneumoniae,Staphylococcus aureus, Staphylococcus epidermidis, Enterococcusfaecalis, Enterococcus faecium, Haemophilus influenzae, Moraxellacatarrhalis, Escherichia coli, Bacteroides fragilis, Bacteroidesthetaiotaomicron, and Clostridium difficile.

In a preferred embodiment, this invention provides compounds of formula(II)

wherein:

R¹ is alkyl;

R² and R³ are independently H, alkyl, or cyanoalkyl, with the provisothat both R² and R³ are not H;

R⁶ is H, alkyl, or hydroxyalkyl;

R⁹, which can be singly or multiply substituted in the ring on the sameor different carbons, is independently selected from the groupconsisting of alkyl, substituted alkyl, alkoxyalkoxy, cycloalkyl,substituted cycloalkyl, alkoxyalkoxy, substituted oxygen, substitutednitrogen, halogen, phenyl, substituted phenyl, —(CH₂)_(n)—OH,—(CH₂)_(n)—NR⁴R⁵, -alkylene-R^(a) where R^(a) is selected frommonofluorophenyl and monochlorophenyl, and branched chain isomersthereof wherein n is an integer of from 1 to 8 inclusive and R⁴ and R⁵are H or alkyl; and

m is 1 or 2; and

prodrugs and pharmaceutically acceptable salts thereof;

with the proviso that the compound of formula II has a minimuminhibition concentration of 32 μg/mL or less against at least one of theorganisms selected from the group consisting of Streptococcuspneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae,Moraxella catarrhalis, Escherichia coli, Bacteroides fragilis,Bacteroides thetaiotaomicron, and Clostridium difficile.

In a particularly preferred embodiment, this invention providescompounds of formula (III):

wherein:

R¹ is alkyl;

R² and R³ are fluoro;

R⁶ is H, alkyl, or hydroxyalkyl;

R⁹, which can be singly or multiply substituted in the ring on the sameor different carbons, is independently selected from the groupconsisting of alkyl, substituted alkyl, alkoxyalkoxy, cycloalkyl,substituted cycloalkyl, alkoxyalkoxy, substituted oxygen, substitutednitrogen, halogen, phenyl, substituted phenyl, —(CH₂)_(n)—OH,—(CH₂)_(n)—NR⁴R⁵, -alkylene-R^(a) where R^(a) is selected frommonofluorophenyl and monochlorophenyl, and branched chain isomersthereof wherein n is an integer of from 1 to 8 inclusive and R⁴ and R⁵are H or alkyl; and

m is 1 or 2; and

prodrugs and pharmaceutically acceptable salts thereof,

with the proviso that the compound of formula III has a minimuminhibition concentration of 32 μg/mL or less against at least one of theorganisms selected from the group consisting of Streptococcuspneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae,Moraxella catarrhalis, Escherichia coli, Bacteroides fragilis,Bacteroides thetaiotaomicron, and Clostridium difficile.

In another preferred embodiment, this invention is directed to acompound of formula (IV):

wherein:

R¹ is alkyl;

R² and R³ are independently H, or alkyl, hydroxy, fluoro, or cyanoalkylor one of R² and R³ is ═NOR⁷ and the other is absent, or one of R² andR³ is ═CH₂ and the other is absent, with the provisos that both R² andR³ are not H; when one of R² and R³ is fluoro, the other is not hydrogenor hydroxy; and when one of R² and R³ is hydroxy, the other is notfluoro, hydrogen, or hydroxy;

R⁶ is selected from the group consisting of —C(O)O-alkylene-cycloalkyl,—C(O)O-alkylene-substituted cycloalkyl, —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-aryl, —C(O)O-substituted aryl, —C(O)O-heteroaryl,—C(O)O-substituted heteroaryl, —[C(O)O]_(p)-alkylene-heterocycle,—[C(O)O]_(p)-alkylene-substituted heterocycle, wherein p is 0 or 1 withthe proviso that —C(O)O-substituted alkyl does not include thefollowing:

R⁷ is H or alkyl;

R⁹, which can be singly or multiply substituted in the ring on the sameor different carbons, is independently selected from the groupconsisting of alkyl, substituted alkyl, alkoxyalkoxy, cycloalkyl,substituted cycloalkyl, alkoxyalkoxy, substituted oxygen, substitutednitrogen, halogen, phenyl, substituted phenyl, —(CH₂)_(n)—OH,—(CH₂)_(n)—NR⁴R⁵, -alkylene-R^(a) where R^(a) is selected frommonofluorophenyl and monochlorophenyl, and branched chain isomersthereof wherein n is an integer of from 1 to 8 inclusive and R⁴ and R⁵are H or alkyl; and

m is 1 or 2; and

prodrugs, tautomers or pharmaceutically acceptable salts thereof;

with the proviso that the compound of formula I has a minimum inhibitionconcentration of 32 μg/mL or less against at least one of the organismsselected from the group consisting of Streptococcus pneumoniae,Staphylococcus aureus, Staphylococcus epidermidis, Enterococcusfaecalis, Enterococcus faecium, Haemophilus influenzae, Moraxellacatarrhalis, Escherichia coli, Bacteroides fragilis, Bacteroidesthetaiotaomicron, and Clostridium difficile.

Lincomycin derivatives within the scope of this invention include thoseset forth in

TABLE I as follows:

Ex. # R¹ R²/R³ R⁶ R⁹* m 1 Methyl H/methyl H ethyl 2 2 Methyl H/methylmethyl propyl 1 3 Methyl H/cyano- methyl propyl 1 methyl 4 Methylhydroxy/ H ethyl 2 methyl 5 Methyl H/hydroxy- methyl propyl 1 imino 6Methyl H/methoxy- methyl propyl 1 imino 7 Methyl H/methyl H butyl 2 8Methyl H/methyl H pentyl 1 9 Methyl H/methyl H isopentyl 1 10 MethylH/methyl H pentyl 1 11 Methyl fluoro/fluoro methyl propyl 1 12 Methylfluoro/fluoro H pentyl 1 13 Methyl H/methyl H 2-(4-fluoro- 1phenyl)ethyl 14 Methyl H/methyl H 3,3-difluoropropyl 1 15 MethylH/methyl H 2-(4-chloro- 1 phenyl)ethyl 16 Methyl H/methyl H2,2-difluoropentyl 1 17 Methyl H/methyl H propyl 2 18 Methyl H/methyl2-hydroxyethyl pentyl 1 19 Methyl H/methyl 2-meth- pentyl 1yl-2-hydroxyethyl 20 Methyl H/methyl 2-meth- pentyl 1 yl-2-hydroxyethyl21 Methyl H/methyl 3-hydroxypropyl n-pentyl 1 22 Methyl H/methyl2-hydroxyethyl isopentyl 1 23 Methyl H/methyl 2-hydroxyethyl3,3-difluoropropyl 1 24 Methyl fluoro/fluoro 2-hydroxyethyl pentyl 1 25methyl H/methyl H pentyl 2 26 methyl H/methyl H methoxy 2 27 methylH/methyl H 1-ethylpropyl 2 28 methyl H/methyl H iso-propyl 2 29 methylH/methyl H butyl 2 30 methyl H/methyl H cyclohexyl 2 31 methyl H/methyl2-hydroxyethyl ethyl 2 32 methyl H/methyl 2-hydroxyethyl pentyl 2 33methyl H/methyl 2-hydroxyethyl propyl 2 34 methyl H/methyl fluorenyl-propyl 2 methyleneoxy- carbonyl 35 methyl H/methyl ethoxycarbonyl propyl2 36 methyl H/methyl phenoxycarbonyl propyl 2 37 methyl H/methyl H4-difluoropentyl 1 38 methyl H/methyl H 3-difluorobutyl 1 39 methylH/methyl 2-hydroxyethyl 3-difluoropentyl 1 40 methyl R2/R3 = H butyl 1vinyl 41 methyl H/methyl H 3-difluoropropyl 2 42 methyl H/methyl H3-difluorobutyl 2 43 methyl H/methyl H 5-difluoropentyl 2 44 methylH/methyl H 5-fluoropentyl 2 45 methyl H/methyl H 4-fluorobutyl 2 46methyl H/methyl H 3-hydroxy-3-ethyl- 2 pentyl 47 methyl H/methyl Hbutoxy 2 48 methyl H/methyl H pentoxy 2 49 methyl H/methyl H4-fluorobutoxy 2 51 methyl H/methyl ethyl ethyl 2 52 methyl H/methyl H3-fluoropropoxy 2 53 methyl H/methyl H 3-trifluoropropoxy 2 54 methylH/methyl H isobutyl 2 55 methyl fluoro/fluoro H propyl 2 56 methylH/methyl H fluoro/propyl 1 57 methyl H/methyl H fluoro/butyl 1 58 MethylH/hydroxy H fluoro/propyl 1 59 methyl H/methyl H 2-methoxyethoxy 60methyl H/methyl H butyl 1 61 methyl H/methyl H 4-difluoropentyl 2 62methyl H/methyl H 3-fluoropropyl 2 63 methyl H/methyl H fluoro/propyl 264 methyl H/methyl H 2-fluoroethoxy 2 65 methyl H/methyl H3-difluoropentyl 1 *Includes R and/or S isomers as either individualisomres or as a mixture

Specific compounds within the scope of this invention include thefollowing compounds:

4-ethyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[3-cyano-2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-ethyl-piperidine-2-carboxylic acid [2-hydroxy-2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-hydroxyimino-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-methoxyimino-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

5-butyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3-methyl-butyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-pentyl-pyrrolidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-[3-(4-fluoro-phenyl)-propyl]-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-propyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-[3-(4-chloro-phenyl)-propyl]-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(2,2-difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-propyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-ethyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(3-hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-ethyl)-4-(3-methyl-butyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-propyl)-1-(2-hydroxy-ethyl)-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-ethyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-pentyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(1-ethyl-propyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-isopropyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-butyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-cyclohexyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-ethyl-1-(2-hydroxy-ethyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-ethyl)-4-pentyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

1-(2-hydroxy-ethyl)-4-propyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylicacid 9H-fluoren-9-ylmethyl ester;

2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylicacid ethyl ester;

2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylicacid phenyl ester;

4-(4,4-difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-butyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-pentyl)-1-(2-hydroxy-ethyl)-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3-difluoro-propyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(4,4-difluoro-butyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yI)-propyl]-amide;

4-(5,5-difluoro-pentyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(5-fluoro-pentyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(4-fluoro-butyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3-ethyl-3-hydroxy-pentyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-butoxy-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-pentyloxy-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(4-fluoro-butoxy)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-butyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-allyl]-amide;

1,4-diethyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3-fluoro-propoxy-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(3,3,3-trifluoro-propoxy)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-isobutyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-propyl-piperidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-fluoro-4-propyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-butyl-4-fluoro-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Fluoro-4-propyl-pyrrolidine-2-carboxylic acid[2-hydroxy-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-(2-methoxyethoxy)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

and prodrugs, tautomers and pharmaceutically acceptable salts thereof.

The compounds, tautomers, prodrugs and pharmaceutically acceptable saltsthereof, as defined herein, may have activity against bacteria,protozoa, fungi, and parasites.

In another aspect, this invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of the compounds defined herein. The pharmaceuticalcompositions of the present invention may further comprise one or moreadditional antibacterial agents, one of which may be active against gramnegative bacteria. One of which may also be active against gram positivebacteria.

In one of its method aspects, this invention is directed to a method forthe treatment of a microbial infection in a mammal comprisingadministering to the mammal a therapeutically effective amount of acompound of this invention. The compound of this invention may beadministered to the mammal orally, parenterally, transdermally,topically, rectally, or intranasally in a pharmaceutical composition.

In another of its method aspects, this invention is directed to a methodfor the treatment of a microbial infection in a mammal comprisingadministering to the mammal a pharmaceutical composition comprising atherapeutically effective amount of a compound of this invention. Thepharmaceutical compositions of the present invention may furthercomprise one or more additional antibacterial agents, one of which maybe active against gram negative bacteria. One of which may also beactive against gram positive bacteria. The pharmaceutical compositionmay be administered to the mammal orally, parenterally, transdermally,topically, rectally, or intranasally.

In a preferred embodiment, the microbial infection being treated is agram positive infection. In an additional embodiment, the infection maybe a gram negative infection. In a further embodiment, the infection maybe a mycobacteria infection, a mycoplasma infection, or a chlamydiainfection.

In yet another aspect, the present invention provides novelintermediates and processes for preparing compounds of formula (I),(II), and (III).

DETAILED DESCRIPTION OF THE INVENTION

As described above, this invention relates to lincomycin derivativesthat exhibit antibacterial activity, in particular gram positiveantibacterial activity. However, prior to describing this invention infurther detail, the following terms will first be defined.

Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below.

“Acyl” means the group —C(O)R′ wherein R′ is alkyl, alkenyl, alkynyl,aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

“Acyloxy” means the group —C(O)OR′, wherein R′ is alkyl, alkenyl,alkynyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

“Alkenyl” means a linear unsaturated monovalent hydrocarbon radical oftwo to eight carbon atoms or a branched monovalent hydrocarbon radicalof three to eight carbon atoms containing at least one double bond,(—C═C—). Examples of alkenyl groups include, but are not limited to,allyl, vinyl, 2-butenyl, and the like.

“Alkoxy” refers to the group “alkyl-O” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

“Alkoxyalkoxy” refers to the group alkyl-O-alkylene-O—, wherein alkyl isas defined herein.

“Alkyl” means a linear saturated monovalent hydrocarbon radical of oneto eight carbon atoms or a branched saturated monovalent hydrocarbonradical of three to eight carbon atoms. Examples of alkyl groupsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-phenyl, and thelike.

“Alkylene” means a linear divalent hydrocarbon radical of one to eightcarbon atoms or a branched divalent hydrocarbon group of three to eightcarbon atoms. Examples of alkylene groups include, but are not limitedto, methylene, ethylene, 2-methylpropylene, and the like.

“Alkylthio” refers to the group “alkyl-S—” which includes, by way ofexample, methylthio, butylthio, and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to eightcarbon atoms or a branched monovalent hydrocarbon radical of three toeight carbon atoms containing at least one triple bond, (—C≡C—).Examples of alkynyl groups include, but are not limited to, ethynyl,propynyl, 2-butynyl, and the like.

“Amino” or “substituted nitrogen” refers to the group “—NR_(a)R_(b)”wherein R^(a) and R_(b) are independently H, alkyl, haloalkyl, alkenyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,or substituted heteroaryl.

“Aminocarboxyalkyl” means a group “—R_(c)C(O)NR_(a)R_(b)” where R^(c) isan alkylene, as defined above, and R_(a) and R_(b) are as defined above.

“Aryl” means a monovalent monocyclic or bicyclic aromatic carbocyclicgroup of six to fourteen ring atoms. Examples include, but are notlimited to, phenyl, naphthyl, and anthryl. The aryl ring may beoptionally fused to a 5-, 6-, or 7-membered monocyclic non-aromatic ringoptionally containing 1 or 2 heteroatoms independently selected fromoxygen, nitrogen, or sulfur, the remaining ring atoms being C where oneor two C atoms are optionally replaced by a carbonyl. Representativearyl groups with fused rings include, but are not limited to,2,5-dihydro-benzo[b]oxepinyl, 2,3-dihydrobenzo[1,4]dioxanyl, chromanyl,isochromanyl, 2,3-dihydrobenzofuranyl, 1,3-dihydroisobenzofuranyl,benzo[1,3]dioxolyl, 1,2,3,4-tetrahydroisoquinolinyl,1,2,3,4-tetrahydroquinolinyl, 2,3-dihydro-1H-indolyl,2,3dihydro-1H-isoindolyl, benzimidazole-2-onyl, 2-H-benzoxazol-2-onyl,and the like.

“Carbonyl” means the group “C(O).”

“Carboxy” means the group “C(O)O.”

“Cyanoalkyl” refers to an alkyl, wherein alkyl is as defined above,substituted with one or more cyano (—CN) groups provided that if twocyano groups are present they are not both on the same carbon atom.Examples of cyanoalkyl groups include, for example, cyanomethyl,2-cyanoethyl, 2-cyanopropyl, and the like.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 20 carbon atomshaving a single or multiple cyclic rings including, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, adamantanyl, and thelike. Cycloalkyl groups of the present invention also include fusedmulticyclic rings wherein one or more of the rings within themulticyclic ring system are aromatic, as long as the point of attachmentto the core or backbone of the structure is on the non-aromatic ring,e.g., fluorenyl.

“Cycloalkylalkyl” means a group —R_(c)R_(d) where R_(c), is an alkylenegroup and R_(d) is a cycloalkyl group, as defined above. Examplesinclude, but are not limited to, cyclopropylmethylene,cyclohexylethylene, and the like.

“Halo” or “Halogen” means fluoro, chloro, bromo, or iodo.

“Haloalkyl” means an alkyl, wherein alkyl is as defined above,substituted with one or more, preferably one to 6, of the same ordifferent halo atoms. Examples of haloalkyl groups include, for example,trifluoromethyl, 3-fluoropropyl, 2,2-dichloroethyl, and the like.

“Heteroaryl” means a monovalent monocyclic or bicyclic aromatic radicalof 5 to 10 ring atoms containing one, two, or three ring heteroatomsselected from N, O, or S, the remaining ring atoms being C.Representative examples include, but are not limited to, thienyl,benzothienyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl,quinoxalinyl, imidazolyl, furanyl, benzofuranyl, thiazolyl, isoxazolyl,benzisoxazolyl, benzimidazolyl, triazolyl, pyrazolyl, pyrrolyl, indolyl,2-pyridonyl, 4-pyridonyl, N-alkyl-2-pyridonyl, pyrazinonyl,pyridazinonyl, pyrimidinonyl, oxazolonyl, and the like.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, from 1 to 10carbon atoms and from 1 to 4 heteroatoms selected from the groupconsisting of nitrogen, sulfur, or oxygen within the ring, wherein, infused ring systems one or more of the rings can be aryl or heteroaryl asdefined herein. Examples of heterocycles and heteroaryls include, butare not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,indoline, phthalimide, 1,2,3,4-tetrahydro-isoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

Heterocycles may be optionally substituted with from one to threesubstituents selected from the group consisting of alkyl, alkenyl,alkynyl, halo, alkoxy, acyloxy, amino, hydroxyl, carboxy, cyano, oxo,nitro, and alkylthio as these terms are defined herein.

“Hydroxy” or “hydroxyl” means the group —OH.

“Hydroxyalkyl” refers to an alkyl, wherein alkyl is as defined abovesubstituted with one or more —OH groups provided that if two hydroxygroups are present they are not both on the same carbon atom. Examplesof hydroxyalkyl groups include, for example, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, and the like.

“Mammal” refers to all mammals including humans, livestock, andcompanion animals.

“Optional” or “optionally” means that the subsequently described eventor circumstance may, but need not, occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “aryl group optionally mono- ordi-substituted with an alkyl group” means that the alkyl may but neednot be present, and the description includes situations where the arylgroup is mono- or disubstituted with an alkyl group and situations wherethe aryl group is not substituted with the alkyl group.

“Pharmaceutically acceptable carrier” means a carrier that is useful inpreparing a pharmaceutical composition that is generally safe, non-toxicand neither biologically nor otherwise undesirable, and includes acarrier that is acceptable for veterinary use as well as humanpharmaceutical use. “A pharmaceutically acceptable carrier” as used inthe specification and claims includes both one and more than one suchcarrier.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include, butare not limited to,

(1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or

(2) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g. an alkali metal ion, an alkalineearth metal ion, or an aluminum ion; or coordinates with an organic basesuch as ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

“Prodrugs” mean any compound which releases an active parent drugaccording to a compound of the subject invention in vivo when suchprodrug is administered to a mammalian subject. Prodrugs of a compoundof the subject invention are prepared by modifying functional groupspresent in a compound of the subject invention in such a way that themodifications may be cleaved in vivo to release the parent compound.Prodrugs include compounds of the subject invention wherein a hydroxy,sulfhydryl or amino group in the compound is bonded to any group thatmay be cleaved in vivo to regenerate the free hydroxyl, amino, orsulfhydryl group, respectively. Examples of prodrugs include, but arenot limited to esters (e.g., acetate, formate, and benzoatederivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxyfunctional groups in compounds of the subject invention, and the like.Specific examples include —C(O)O-alkylene-cycloalkyl,—C(O)O-alkylene-substituted cycloalkyl, —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-aryl, —C(O)O-substituted aryl, —C(O)O-heteroaryl,—C(O)O-substituted heteroaryl, —[C(O)O]_(p)-alkylene-heterocycle,—[C(O)O]_(p)-alkylene-substituted heterocycle, wherein p is 0 or 1 withthe proviso that —C(O)O-substituted alkyl does not include thefollowing:

“Substituted alkyl” means an alkyl group, as defined above, in which oneor more of the hydrogen atoms has been replaced by a halogen (i.e., Cl,Br, F, or I), oxygen, hydroxy, amine (primary), amine (secondary-alkylsubstituted by alkyl above) amine (tertiary-alkyl substituted by alkylas above), sulfur, —SH, or phenyl. Examples of substituted alkyl groupsinclude, but are not limited to, 1-fluoroethyl, 1-chloroethyl,2-fluoroethyl, 2-chloroethyl, 1-bromopropyl, 2-iodopropyl,1-chlorobutyl, 4-flurobutyl, and 4-chlorobutyl.

“Substituted aryl” means an aryl ring substituted with one or moresubstituents, preferably one to three substituents selected from thegroup consisting of alkyl, alkenyl, alkynyl, halo, alkoxy, acyloxy,amino, hydroxy, carboxy, cyano, nitro, alkylthio, and thioalkyl. Thearyl ring may be optionally fused to a 5-, 6-, or 7-membered monocyclicnon-aromatic ring optionally containing 1 or 2 heteroatoms independentlyselected from oxygen, nitrogen, or sulfur, the remaining ring atomsbeing carbon where one or two carbon atoms are optionally replaced by acarbonyl.

“Substituted cycloalkyl” means a cycloalkyl substituted with an alkylgroup, wherein alkyl is as defined above or a group as defined above forsubstituted alkyl. Representative examples include, but are not limitedto, 2-cyclopropylethyl, 3-cyclobutylpropyl, 4-cyclopentylbutyl,4-cyclohexylbutyl, and the like.

“Substituted heteroaryl” means a heteroaryl ring, wherein heteroaryl isas defined above, substituted with one or more substituents, preferablyone to three substituents selected from the group consisting of alkyl,alkenyl, alkynyl, halo, alkoxy, acyloxy, amino, hydroxy, carboxy, cyano,nitro, alkylthio, and thioalkyl, wherein said substituents are asdefined herein.

“Substituted oxygen” refers to the group “—O—R^(d)” wherein R^(d) isalkyl, haloalkyl, alkenyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, or substituted heteroaryl, wherein saidsubstituents are as defined herein.

“Substituted phenyl” means a phenyl ring wherein one or more of thehydrogen atoms has been replaced by a halogen, hydroxy, alkyl, amine(primary, secondary, and tertiary with the latter two alkylsubstituted), —SH, and phenyl. Representative examples include, but arenot limited to, p-bromophenyl, m-iodophenyl, o-chlorophenyl,p-ethylphenyl, m-propylphenyl, o-methylphenyl, and p-octylphenyl.

“Thioalkyl” refers to an alkyl, wherein alkyl is as defined above,substituted with one or more —SH groups provided that if two hydroxygroups are present they are not both on the same carbon atom. Examplesof thioalkyl groups include, for example, thiomethyl, 2-thioethyl,2-thiopropyl, and the like.

“Therapeutically effective amount” means the amount of a compound orcomposition that, when administered to a mammal for treating a disease,is sufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compoundor composition, the disease and its severity and the age, weight, etc.,of the mammal to be treated.

“Treating” or “treatment” of a disease includes:

(1) preventing the disease, i.e. causing the clinical symptoms of thedisease not to develop in a mammal that may be exposed to or predisposedto the disease but does not yet experience or display symptoms of thedisease,

(2) inhibiting the disease, i.e., arresting or reducing the developmentof the disease or its clinical symptoms, or

(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

“Tautomer” refers to an isomer in which migration of a hydrogen atomresults in two or more structures.

The compounds of the present invention are generally named according tothe IUPAC or CAS nomenclature system. Abbreviations that are well knownto one of ordinary skill in the art may be used (e.g. “Ph” for phenyl,“Me” for methyl, “Et” for ethyl, “Bn” for benzyl, “h” for hour and “rt”for room temperature).

General Synthetic Schemes

Compounds of this invention can be made by the methods depicted in thereaction schemes shown below.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Toronto ResearchChemicals (North York, ON Canada), Aldrich Chemical Co. (Milwaukee,Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemie, or Sigma (St.Louis, Mo., USA) or are prepared by methods known to those skilled inthe art following procedures set forth in references such as Fieser andFieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley andSons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989), Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition), and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Theseschemes are merely illustrative of some methods by which the compoundsof this invention can be synthesized, and various modifications to theseschemes can be made and will be suggested to one skilled in the arthaving referred to this disclosure.

As it will be apparent to those skilled in the art, conventionalprotecting groups may be necessary to prevent certain functional groupsfrom undergoing undesired reactions. Suitable protecting groups forvarious functional groups, as well as suitable conditions for protectingand deprotecting particular function groups are well known in the art.For example, numerous protecting groups are described in T. W. Greeneand G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition,Wiley, N.Y., 1991, and references cited therein.

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography, and the like. Such materials may be characterized usingconventional means, including physical constants and spectral data.

The compounds of this invention will typically contain one or morechiral centers. Accordingly, if desired, such compounds can be preparedor isolated as pure stereoisomers. All such stereoisomers (and enrichedmixtures) are included within the scope of this invention, unlessotherwise indicated. Pure stereoisomers (or enriched mixtures) may beprepared using, for example, optically active starting materials orstereoselective reagents well-known in the art. Alternatively, racemicmixtures of such compounds can be separated using, for example, chiralcolumn chromatography, chiral resolving agents, and the like.

Preparation of Compounds of Formula (I)

In general, to prepare the compounds of formula (I) of the presentinvention, an appropriately 7-substititued lincosamine intermediate andan appropriately substituted pyrrolidinyl or piperidyl carboxylic acidare condensed under reactive conditions, preferably in an inert organicsolvent, in the presence of a coupling reagent and an organic base. Thisreaction can be performed with any number of known coupling reagents,such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), 1-hydroxybenzotriazole hydrate (HOBT) withcarbodiimides, isobutyl chloroformate, diphenylphosphoryl azide (DPPA),and the like. Suitable organic bases include diisopropylethylamine(DIEA), triethylamine (TEA), pyridine, N-methyl morpholine, and thelike. Suitable inert organic solvents which can be used include, forexample, N,N-dimethylformamide, acetonitrile, dichloromethane, and thelike. This reaction is typically conducted using an excess of carboxylicacid to lincosamine at temperatures in the range of about 0° C. to about50° C. The reaction is continued until completion, which typicallyoccurs in from about 2 to 12 h.

Appropriately 7-substititued lincosamine intermediates, as defined inthe present invention (i.e., R²/R³), are synthesized by methods wellknown to those of skill in the art from methyl6-amino-6,8-dideoxy-1-thio-erythro-α-D-galacto-octopyranoside, which canbe prepared as described by Hoeksema, et al., Journal of the AmericanChemical Society, 1967, 89 2448–2452. Illustrative syntheses for7-substituted lincosamine intermediates are shown below in Schemes 1–5.

Appropriately substituted pyrrolidinyl or piperidyl carboxylic acidintermediates, as defined in the present invention (i.e., R⁹), are alsosynthesized by methods well known to those of skill in the art fromprolines and pyridines. The prolines and pyridines that can be used inthe synthesis of the carboxylic acid intermediates of the presentinvention include, for example, 4-oxoproline and 4-substitutedpyridines. The prolines and pyridines used in the synthesis arecommercially available from vendors such as Aldrich and Sigma.Alternatively, these prolines and pyridines can be prepared by methodswell known in the art. Illustrative syntheses for appropriatelysubstituted pyrrolidinyl or piperidyl carboxylic acid intermediates areshown below in Schemes 6–10.

Scheme 1 below illustrates a general synthesis of a lincosamineintermediate 1c wherein P is an N-protecting group, preferably eitherCbz or Boc, and R¹ is as defined for formula (I).

Scheme 1. General Synthesis of Lincosamine Intermediate 1c (a)N-Protection (Boc, Cbz); (b) O-silyl Protection (TMS); (c) SwernOxidation

As shown in Scheme 1, methyl6-amino-6,8-dideoxy-1-thio-erythro-α-D-galacto-octopyranoside, 1a, isprepared as described by Hoeksema, et al., Journal of the AmericanChemical Society, 1967, 89 2448–2452. The amino functional group and thehydroxy functional groups of the product 1a are then protected withsuitable protecting groups. Suitable N-protecting groups can be formedby the addition of di-t-butyldicarbonate, N-(benzyloxycarbonyloxy)succinimide, and the like. The hydroxy groups can be protected as silylethers. The hydroxyl group can be converted to trimethylsilyl (TMS)ethers by reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide inthe presence of an appropriate organic base such as triethylamine (TEA)or trimethylsilyl chloride in the presence of an organic base such astriethylamine. The N-protection is typically accomplished before theO-protection. Chromatography of the crude product on silica afterevaporation of the solvent provides the protected product 1b.

The 7-O-trimethylsilyl group of 1b is chemoselectively deprotected andoxidized to provide the 7-keto-lincosamine derivative 1c. This selectivetransformation is performed by addition of the protected product 1b todimethylsulfoxide and oxalyl chloride in an inert organic solvent suchas dichloromethane followed by an appropriate organic base such astriethylamine. Alternatively, the transformation may be performed byaddition of 1b to dimethyl sulfoxide and an appropriate activating agentsuch as trifluoroacetic anhydride in an inert organic solvent. Thereaction is typically conducted at temperatures in the range ofapproximately −70° C. to −80° C. The resulting reaction mixture isstirred at the low temperature and is then allowed to warm toapproximately −50° C. The reaction is maintained at this secondtemperature for approximately 1 h to 3 h. To the reaction mixture isadded a suitable organic base, such as TEA, pyridine, and the like. Thereaction mixture is appropriately worked up to provide the product 1c.The general class of conditions used in the transformation of 1b to 1cis known in the art as Swern oxidation conditions.

Scheme 2 below illustrates a general synthesis of a lincosamineintermediate 2b wherein P is an N-protecting group, preferably eitherCbz or Boc, R¹ is as defined for formula (I), and one of R² and R³ ishydrogen and the other is as defined for formula (I).

Scheme 2. General Synthesis of Lincosamine Intermediate 2b (a) WittigOlefination (R²PPh₃ ⁺X⁻, R²PO(OEt)₂, Base, Solvent); (b) and (c) H₂/Pd,Global De-protection

As shown in Scheme 2, a keto-lincosamine intermediate 1c is reacted toform an alkene using the Wittig or Homer-Wadsworth-Emmons reaction. Inthis reaction, a suitable phosphonium salt or phosphonate isdeprotonated using a strong base to form a phosphorus ylide. Suitablephosphonium salts which can be used are alkyltriphenylphosphoniumhalides, which can be prepared by the reaction of triphenylphosphine andan alkyl halide. Suitable phosphorous compounds include, for example,methyltriphenylphosphonium bromide, diethyl(cyanomethyl)phosphonate andthe like. Suitable strong bases which can be used to form the ylideinclude organolithium reagents, potassium tert-butoxide, and the like.The formation of the phosphorus ylide is typically conducted under aninert atmosphere, such as N₂, in an inert organic solvent such astoluene, THF, and the like, at low temperatures.

After formation of the phosphorus ylide, the product 1c is added to thereaction. The reaction conveniently can be performed at temperaturesbetween −40° C. and room temperature and is stirred until completion,typically 1 to 4 h. The resulting organic solution is worked-up andchromatography of the crude product on silica provides the alkeneproduct 2a.

Optionally, the product of 2a may be purified using conventionaltechniques, such as chromatography and said purified product may be usedin the subsequent coupling reaction to yield vinyl lincosaminederivatives of the present invention.

The product 2a is then hydrogenated to provide the saturated product 2b.The hydrogenation is typically performed in a polar organic solvent suchas methanol, ethanol, and the like, using 10% palladium on carbon in aParr bottle. The bottle is purged, and charged with H₂ to approximately50 to 70 psi and shaken until completion, typically approximately 12 to24 h. The resulting reaction mixture is filtered, e.g. through celite,and rinsed with a polar organic solvent such as methanol. The organicsolution is worked up by transferring to a resin funnel containing dry,washed Dowex™ 50w400x H⁺ form and shaken. After washing the resin withmethanol and water, the product 2b is eluted from the resin by washingwith 5% TEA in MeOH. The product can also be purified by silica gelcolumn chromatography.

Scheme 3 illustrates a general synthesis of a lincosamine intermediate3b wherein P is an N-protecting group, preferably either Cbz or Boc, R¹is as defined for formula (I), and one of R² or R³ is alkyl and theother is —OH.

Scheme 3. General Synthesis of Lincosamine Intermediate 3b (a) R²M(Carbon Nucleophile); (b) (i) TMS De-protection (H⁺ or F⁻) and (ii)N-deprotection

As shown in Scheme 3, suitable carbon nucleophiles add to7-ketolincosamine intermediate 1c in suitable inert organic solvents toprovide a 7-hydroxy lincosamine intermediate 3b. Suitable carbonnucleophiles include methylmagnesium chloride, diethyl zinc, sodiumacetylide and the like and suitable inert organic solvents which can beused include THF, diethyl ether, toluene, and the like. The reaction istypically conducted at reduced temperatures, approximately at 0° C., forabout 3 to 5 h. The reaction is then quenched with a saturated aqueousacidic solution, such as saturated aqueous NH₄Cl/H₂O. The quenchedmixture is then worked up and can be purified by chromatography toprovide the product 3b.

Scheme 4 below illustrates a general synthesis of a lincosamineintermediate 4b wherein P is a N-protecting group, preferably Boc, R¹ isas defined for formula (I), and R²/R³ is an oxime (═NOR⁷), wherein R⁷ isas defined for formula (I).

Scheme 4. General Synthesis of 7-oxime-lincosamines 4b

As shown in Scheme 4, the lincosamine intermediate 1c is converted tothe oxime by stirring in the presence of a suitable reagent such asO-trimethylsilylhydroxylamine, O-alkylhydroxylamine hydrochloride (forexample, O-methylhydroxylamine hydrochloride), and the like. Thereaction is typically conducted in a polar organic solvent such asmethanol. The reaction conveniently can be conducted at rt inapproximately 8 to 24 h. The solvent is removed to provide theN-protected product 4a.

Removal of the protecting group can be carried out with acids, such astrifluoroacetic acid (TFA), hydrochloric acid, p-toluenesulfonic acid,and the like, in an inert organic solvent such as dichloromethane,dichloroethane, dioxane, THF, and the like. The removal is typicallyconducted at low temperatures, e.g., 0° C., and then gradually allowedto warm to room temperature to provide the product 4b.

Scheme 5 below illustrates a general synthesis of a lincosamineintermediate 5b wherein R² and R³ are both fluorine, P is anN-protecting group, preferably Cbz or Boc, and R¹ is as defined forformula (I).

Scheme 5. General Synthesis of 7-deoxy-7,7-difluorolincosamines 5b (a)F⁻; (b) Ac₂O, Pyridine, DMAP; (c) DAST; (d) TFA

As shown in Scheme 5, the lincosamine intermediate 1c is contacted witha suitable fluoride in an inert organic solvent. Suitable fluorideswhich can be used include tetrabutylammonium fluoride, Amberlite resinA-26 F form, HF.pyridine and the like. Suitable inert organic solventsinclude THF, acetonitrile, dichloromethane, dioxane, and the like. Thereaction conveniently can be conducted at rt in about 1 to 2 h. Theproduct (not shown) can be purified on a silica gel column.

The O-protecting groups on the product obtained from the column areconverted by contact with acetic anhydride and dimethylaminopyridine(DMAP) in a suitable mixture of an inert organic solvent and an organicbase, such as, for example, dichloromethane and pyridine. The reactionconveniently can be conducted at rt in approximately 6 to 12 h. Theproduct can be purified on silica gel column to provide product 5a.

The product 5a is contacted with a suitable fluorinating reagent andthen the N-protecting group is removed to provide the product 5b.Suitable fluorinating reagents which can be used include, for example,dimethylaminosulfurtrifluoride,[bis(2-methoxyethyl)amino]sulfurtrifluoride, and the like. The reactionis typically conducted in an inert organic solvent such asdichloromethane, ethylacetate, THF, and the like at room temperature inapproximately 6 to 12 h.

Removal of the protecting group can be carried out with acids, such astrifluoroacetic acid (TFA), hydrochloric acid, p-toluenesulfonic acid,and the like, in an inert organic solvent such as dichloromethane,dichloroethane, dioxane, THF, and the like. The removal is typicallyconducted at low temperatures, e.g., 0° C., and then gradually allowedto warm to room temperature to provide the product 5b.

Scheme 6 below illustrates a general synthesis of a proline intermediate6c wherein R⁹ is as defined for formula (I).

Scheme 6. General Synthesis of cis/trans R⁹-proline IntermediateMixtures 6c (a) R⁹P⁺Ph₃Br⁻, NaH, DMSO; (b) H₂/Pt

As shown in Scheme 6, the product 6c is prepared as described inMagerlein, Journal of Medicinal Chemistry 1972, 15, 1255–1259. Compound6a is commercially available from vendors such as RSP (ScientificResearch Consortium, Inc.). Alternatively, 6a can be prepared fromcommercially available protected hydroxy prolines by methods well knownin the art. See, e.g., Demange, et al., Tetrahedron Letters 1998,39,1169–1172.

Scheme 7 below illustrates a general synthesis of trans-R⁹-prolineintermediates 7d, wherein R⁹ is alkyl or substituted alkyl.

Scheme 7. General Synthesis of trans-alkylprolines 7d (a) (i) LiHMDS,THF −78° C., (ii) bromoalkene; (b) (i) LiBHEt₃, THF −78° C., (ii)BF₃OEt₂, Et₃SiH; (c) H₂ Pd/C

As shown in Scheme 7, a protected 4-oxoproline, 7a, is enolated with asuitable enolation agent and then alkylated with a suitable alkylatingagent in an inert organic solvent to provide a lactam 7b (wherein R^(9′)is alkenyl), as described in the literature procedure by Zhang, et al.,J.A.C.S. 1998,120 3894–3902. Compound 7a is commercially available fromvendors such as Bachem. Alternatively, 7a can be prepared by methodswell known in the art. Suitable enolating agents include LiHMDS,LiN(iPr)₂, and the like, and suitable alkylating agents include allylicand benzylic bromides, for example, 4-bromo-2-methyl-2-butene andcis-1-bromo-2-pentene, allylbromide, and the like.

The lactam 7b is reduced using a suitable reducing agent to provide apyrrolidine 7c, wherein R^(9′) is alkenyl. The reduction is preformed bya two-step sequence involving superhydride reduction of the lactam tothe hemiaminal and the subsequent reduction of the hemiaminal. Suitablereducing agents which can be used include Et₃SiH/BF₃.OEt₂, Et₃SiH/TiCl₄,and the like.

The pyrrolidine 7c is then hydrogenated to simultaneously remove theunsaturation in the R^(9′) substituent and remove the benzyl protectinggroup from the carboxylic acid to provide the product 7d. Thehydrogenation is typically performed in a polar organic solvent such asmethanol, ethanol, and the like, using 10% palladium on carbon in a Parrbottle. The bottle is purged, and charged with H₂ to approximately 50 to70 psi and shaken until completion, typically approximately 5 to 24 h.The reaction mixture is filtered, e.g., through a celite pad, and washedwith a polar organic solvent, such as methanol. Evaporation of thecombined washings and filtrate affords the product 7d, wherein R⁹ is analkyl or substituted alkyl.

Scheme 8 below illustrates a general synthesis of trans-R⁹-prolineintermediates 8c, wherein R⁹ is alkyl or substituted alkyl.

Scheme 8. General Synthesis of trans-R⁹-substituted Prolines 8c, WhereinR⁹ is Alkyl or substituted Alkyl (a) O₃, DCM, −78° C., DMS; (b) P⁺Ph₃salt, Base; (c) H₂, Pd/C

As shown in Scheme 8, the product 7d is ozonolized to provide thealdehyde 8a. The ozonolysis reaction is typically conducted in ananhydrous inert organic solvent, such as dichloromethane, dioxane, THF,and the like, at low temperatures, e.g., −78° C. followed by quenchingof the reaction with a reducing agent such as DMS, Ph₃P.

The aldehyde, 8a, is reacted with a suitable phosphonium salt in thepresence of a strong base in an inert organic solvent. Suitablephosphonium salts which can be used include, for example, fluorobenzylphosphonium chloride, 4-chlorobenzyl phosphonium chloride,dibromofluoromethane and triphenylphosphine, and the like. Suitablebases which can be used include potassium t-butoxide, organolithiumreagents, and activated zinc. Suitable organic solvents which can beused include toluene, THF, dimethylacetamide, and the like. The reactionis typically conducted in an inert atmosphere, such as under nitrogen,with vigorous stirring. The reaction is typically conducted at rt toapproximately 110° C. for 1 to 2 h. The resulting reaction mixture isappropriately worked-up and can be purified by chromatography to provide8b (wherein R^(9′) is alkenyl).

The product 8b is then hydrogenated to provide the product 8c. Thehydrogenation is typically performed in a polar organic solvent such asmethanol, ethanol, and the like, using 10% Palladium on carbon in a Parrbottle. The bottle is purged, and charged with H₂ to approximately 40 to70 psi and shaken until completion, typically approximately 4 to 24 h.The reaction mixture is filtered, e.g., through a celite pad and washedseveral times with a polar organic solvent, such as methanol.Evaporation of the combined washings and filtrate affords the product8c, wherein R⁹ is an alkyl or substituted alkyl.

Scheme 9 below illustrates a general synthesis of trans-R⁹-prolineintermediates 9d, wherein R⁹ is substituted alkyl wherein X is halo.

Scheme 9. Example Synthesis of Trans-halosubstituted Alkyl Prolines 9d(a) Tetraallyltin, BF₃.Et₂O; (b) DMSO, (COCl)₂, TEA; (c) DAST (d)10%Pd/C, H₂

As shown in Scheme 9, aldehyde, 8a, is reduced and alkylated using asuitable reagent in an inert organic solvent to provide a hydroxyalkenylsubstituted proline, 9a. Suitable reagents to reduce and alkylate thealdehyde include tetraallyltin/boron trifluoride etherate,allylTMS/boron trifluoride etherate and suitable inert organic solventswhich can be used include THF, dichloromethane, and the like. Thereaction is typically conducted at low temperatures, e.g., 0° C., forapproximately 1 to 2 h. To the reaction mixture is added a solution of asuitable fluoride salt in water, for example potassium fluoride inwater, followed by the addition of methanol. The reaction mixture isfiltered, for example, over celite. The product can be purified bychromatography to provide 9a.

The hydroxyalkenyl substituted proline, 9a, is oxidized to the ketone bycontact with a suitable oxidizing agent in an inert organic solvent.Suitable oxidizing agents include oxalyl chloride/DMSO, Dess Martinperiodinane, and the like. Suitable inert organic solvents includedichloromethane, and the like. The reaction is typically conducted atreduced temperatures, e.g., −72° C. to −50° C., for approximately 30 minto 2 h. To the reaction mixture is added a suitable organic base, suchas triethylamine. The reaction mixture is worked up to provide product9b.

The keto-substituted product 9b is halogenated by contact with asuitable halogenating agent in an inert organic solvent. Suitablehalogenating agents which can be used include diethylaminosulfurtrifluoride, [bis(2-methoxyethyl)amino] sulfur trifluoride, and thelike. Suitable inert organic solvents which can be used includedichloromethane, ethyl acetate, THF, and the like. The reaction istypically conducted at low temperatures in the range of approximately−30° C. to −78° C. The reaction mixture is gradually allowed to warm tort and stirred at rt until completion, typically in 6 to 12 h. Thereaction mixture is worked up and can be purified by chromatography toprovide 9c.

The product 9c is then hydrogenated to provide the product 9d. Thehydrogenation is typically performed in a polar organic solvent such asmethanol, ethanol, and the like, using 10% palladium on carbon in a Parrbottle. The bottle is purged, and charged with H₂ to approximately 40 to70 psi and shaken until completion, typically approximately 4 to 24 h.The reaction mixture is filtered, e.g., through a celite pad and washedseveral times with a polar organic solvent, such as methanol.Evaporation of the combined washings and filtrate affords the product9d.

Scheme 10 below illustrates a general synthesis, as described in Shuman,Journal of Organic Chemistry. 1990, 55, 741–750, of substituted pyridinecarboxylic acid intermediates 10b, wherein R⁹ is as defined for formula(I).

Scheme 10. General Synthesis of Substituted pyridin-2-yl CarboxylicAcids 10b

As shown in Scheme 10, an appropriately substituted pyridine, iscontacted with a suitable oxidizing agent in an inert organic solvent.The appropriately substituted pyridine starting materials arecommercially available from vendors such as Aldrich and Sigma.Alternatively, these pyridines can be prepared by methods well known inthe art. Suitable oxidizing agents which can be used include hydrogenperoxide, MCPBA, and the like. The reaction is typically conducted atreflux for 6 to 12 h. The reaction mixture is then contacted with asuitable cyanide reagent to provide the cyano-substituted pyridine, 10a.Suitable cyanide reagents which can be used include trimethylsilylcyanide, HCN, and the like. Suitable inert organic solvents includedichloromethane, dioxane, THF, and the like. The reaction convenientlycan be conducted at rt in approximately 6 to 12 h. The reaction mixtureis worked up to provide the cyano-substituted pyridine, 10a.

The cyano-substituted pyridine, 10a, is then hydrolyzed to provide thepyridin-2-yl carboxylic acid 10b by contact with a suitable acid.Suitable acids for hydrolyzing the cyano group to the carboxylic acidinclude hydrochloric acid, aqueous sulfuric acid, and the like. Thereaction is typically conducted at reflux in 6 to 12 h.

Scheme 11 below illustrates the coupling reaction of a lincosamineintermediate, prepared as described above in Schemes 1–5, and apyrrolidinyl or piperidinyl carboxylic acid, prepared as described abovein Schemes 6–10, wherein R¹, R², R³, R6, and R⁹ are as defined forformula (I) and P¹ is a suitable O-protecting group and P² is a suitableN-protecting group.

Scheme 11. General Coupling and Deprotection Methods

As shown in Scheme 11, an appropriately 7-substititued lincosamineintermediate (prepared, for example, according to any one of Schemes1–5) and an appropriately substituted pyrrolidinyl or piperidylcarboxylic acid (prepared, for example, according to any one of Schemes6–10) are condensed under reactive conditions, preferably in an inertorganic solvent, in the presence of a coupling reagent and an organicbase. This reaction can be performed with any number of known couplingreagents, such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluroniumhexafluorophosphate (HATU), 1-hydroxybenzotriazole hydrate (HOBT) withcarbodiimides, diphenylphosphoryl azide (DPPA), isobutyl chloroformate,and the like. Suitable organic bases include diisopropylethylamine(DIEA), triethylamine (TEA), pyridine, N-methyl morpholine, and thelike. Suitable inert organic solvents which can be used include, forexample, N,N-dimethylformamide, acetonitrile, dichloromethane, and thelike. This reaction is typically conducted using an excess of carboxylicacid to lincosamine at temperatures in the range of about 0° C. to about50° C. The reaction is continued until completion, which typicallyoccurs in from about 2 to 12 h.

Removal of the protecting groups can be carried out with acids, such astrifluoroacetic acid (TFA), hydrochloric acid, p-toluenesulfonic acid,and the like, in an inert organic solvent such as dichloromethane,dichloroethane, dioxane, THF, and the like. The removal is typicallyconducted at low temperatures, e.g., 0° C., and then gradually allowedto warm to room temperature to provide the product.

Also as shown in Scheme 11, an appropriately 7-substititued lincosamineintermediate (prepared, for example, according to any one of Schemes1–5) and an appropriately substituted pyridin-2-yl carboxylic acid(prepared, for example, according to Scheme 10) are condensed underreactive conditions, preferably in an inert organic solvent, in thepresence of a coupling reagent and an organic base, as described above.

The pyridine 11b is hydrogenated to provide the piperidyl product. Thehydrogenation is typically performed in a polar organic solvent such asmethanol, ethanol, and the like, using platinum(IV)oxide in the presenceof an acid such as HCl, acetic acid, and the like, in a Parr bottle. Thebottle is purged, and charged with H₂ to approximately 40 to 70 psi andshaken until completion, typically approximately 24 h. The reactionmixture is filtered, e.g., through a celite pad, and washed severaltimes with a polar organic solvent such as methanol. Evaporation of thecombined washings and filtrate affords the piperidyl product.

The coupling of pyridine carboxylic acids and lincosamines to pyridine11b followed by reduction to the piperidyl product may also be conductedas described in Birkenmeyer, et al., Journal of Medicinal Chemistry1984, 27, 216–223.

Scheme 12 below illustrates the alkylation of the nitrogen of thepyrrolidinyl or piperidinyl ring, wherein R⁶ is alkyl, hydroxyalkyl,alkylene-substituted heterocycle, or alkylene-heterocycle, and R¹, R²,R³, and R⁹ are as defined for formula (I).

Scheme 12. General Synthesis of 1′-N-substituted Lincosamines. a.Alkylating Agents

As shown in Scheme 12, the lincosamine 12a can be N-substituted bycontact with an alkylating agent in the presence of a suitable base toprovide a product 12b. Suitable alkylating agents which can be usedinclude epoxides, alkyl bromides, and the like. Suitable bases which canbe used include potassium carbonate, cesium carbonate triethylamine, andthe like. The alkylation reaction is typically conducted in a polarorganic solvent such as methanol or DMF. The alkylation reaction istypically conducted at low temperatures in the range of 0° C. to −10° C.for 10 to20 h.

Scheme 13 below illustrates the acylation of the nitrogen of thepyrrolidinyl or piperidinyl ring, wherein R⁶ is—C(O)O-alkylene-cycloalkyl, —C(O)O-alkylene-substituted cycloalkyl,—C(O)O-alkyl, —C(O)O-substituted alkyl, —C(O)O-aryl, —C(O)O-substitutedaryl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl,—[C(O)O]-alkylene-heterocycle, —[C(O)O]-alkylene-substitutedheterocycle, and R¹, R², R³, and R⁹ are as defined for formula (I).

Scheme 13. General Synthesis of 1′-N-substituted Lincosamines

As shown in Scheme 13, the lincosamine 12a can be N-substituted bycontact with an acyl chloride 101, such as R⁶—X, wherein X is a suitableleaving group, and is preferably halogen, even more preferably chloridein the presence of a suitable base to provide a product 102. Examples ofcompound 101, include bromofluorenyl, Cl—C(O)O-alkyl, Cl—C(O)O-aryl, andthe like. Suitable bases which can be used include DCC, TEA, and thelike. The reaction is typically conducted in a polar organic solventsuch as methanol or DMF. The reaction is typically conducted at lowtemperatures in the range of −10° C. to 20° C.

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of the subject inventionare usually administered in the form of pharmaceutical compositions.These compounds can be administered by a variety of routes includingoral, parenteral, transdermal, topical, rectal, and intranasal. Thesecompounds are effective as both injectable and oral compositions. Suchcompositions are prepared in a manner well known in the pharmaceuticalart and comprise at least one active compound.

This invention also includes pharmaceutical compositions that contain,as the active ingredient, one or more of the compounds of the subjectinvention above associated with pharmaceutically acceptable carriers. Inmaking the compositions of this invention, the active ingredient isusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be in the form of a capsule, sachet,paper or other container. The excipient employed is typically anexcipient suitable for administration to human subjects or othermammals. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The quantity of active component, that is the compound according to thesubject invention, in the pharmaceutical composition and unit dosageform thereof may be varied or adjusted widely depending upon theparticular application, the potency of the particular compound and thedesired concentration.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Preferably, the compound of the subject invention above isemployed at no more than about 20 weight percent of the pharmaceuticalcomposition, more preferably no more than about 15 weight percent, withthe balance being pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically or therapeuticallyeffective amount. It will be understood, however, that the amount of thecompound actually administered will be determined by a physician, in thelight of the relevant circumstances, including the condition to betreated, the severity of the bacterial infection being treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

In therapeutic use for treating, or combating, bacterial infections inwarm-blooded animals, the compounds or pharmaceutical compositionsthereof will be administered orally, topically, transdermally, and/orparenterally at a dosage to obtain and maintain a concentration, thatis, an amount, or blood-level of active component in the animalundergoing treatment which will be antibacterially effective. Generally,such antibacterially or therapeutically effective amount of dosage ofactive component (ie., an effective dosage) will be in the range ofabout 0.1 to about 100, more preferably about 1.0 to about 50 mg/kg ofbody weight/day.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerthat serves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner.

The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/capsule) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 mg  Starch 45.0mg  Microcrystalline cellulose 35.0 mg  Polyvinylpyrrolidone 4.0 mg (as10% solution in sterile water) Sodium carboxymethyl starch 4.5 mgMagnesium stearate 0.5 mg Talc 1.0 mg Total  120 mg 

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch and magnesium stearate are blended, passedthrough a No. 20 mesh U.S. sieve, and filled into hard gelatin capsulesin 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient 25 Saturated fatty acid glyceridesto 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mgSucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 8

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

Formulation Example 9

A subcutaneous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1–10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Formulation Example 11

An intravenous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient  250 mg Isotonic saline 1000 mg

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half-life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half-life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each ofwhich is incorporated herein by reference.

As noted above, the compounds administered to a patient are in the formof pharmaceutical compositions described above. These compositions maybe sterilized by conventional sterilization techniques, or may besterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile aqueous carrier prior to administration. The pH of thecompound preparations typically will be between 3 and 11, morepreferably from 5 to 9 and most preferably from 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

In general, the compounds of the subject invention will be administeredin a therapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ ED₅₀.Compounds that exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range which includes the IC₅₀ (the concentration of thetest compound which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Utility

The compounds, prodrugs and pharmaceutically acceptable salts thereof,as defined herein, may have activity against a variety of bacteria,protozoa, fungi, and parasites. By way of example, the compounds,prodrugs and pharmaceutically acceptable salts thereof may be activeagainst gram positive and gram negative bacteria. The compounds,prodrugs and pharmaceutically acceptable salts thereof may be activeagainst a variety of fungi, including fungi from the genus Mucor andCandida, e.g., Mucor racemosus or Candida albicans. The compounds,prodrugs and pharmaceutically acceptable salts thereof may be activeagainst a variety of parasites, including malaria and cyptosporidiumparasite.

The compounds of the subject invention exhibit activity against avariety of bacterial infections, including, for example, gram positiveinfections, gram negative infections, mycobacteria infections,mycoplasma infections, and chlamydia infections.

Since the compounds of the subject invention exhibit potent activities avariety of bacteria, such as gram positive bacteria, the compounds ofthe present invention are useful antimicrobial agents and may beeffective against a number of human and veterinary pathogens, includinggram positive bacteria. The Gram positive organisms against which thecompounds of the present invention are effective include Streptococcuspneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae,Moraxella catarrhalis, Escherichia coli, Bacteroides fragilis,Bacteroides thetaiotaomicron, and Clostridium difficile, and the like.

The compounds of the subject invention may be combined with one or moreadditional antibacterial agents. One or more of the additionalantibacterial agents may be active against gram negative bacteria.Additionally, one or more of the additional antibacterial agents may beactive against gram positive bacteria. The combination of the compoundsof the subject invention and the one or may additional antibacterialagents may be used to treat a gram negative infection. Additionally, thecombination of the compounds of the subject invention and the one ormore additional antibacterial agents may be used to treat a grampositive infection. The combination of compounds of the subjectinvention and the one or more additional antibacterial agents may alsobe used to treat a mycobacteria infection, mycoplasma infection, orchlamydia infection.

The in vitro activity of compounds of the subject invention may beassessed by standard testing procedures such as the determination ofminimum inhibitory concentration (MIC) by agar dilution as described in“Approved Standard. Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria that Grow Aerobically,” 3^(rd) ed., published 1993 bythe National Committee for Clinical Laboratory standards, Villanova,Pa., USA.

The amount administered to the mammalian patient will vary dependingupon what is being administered, the purpose of the administration, suchas prophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from a disease in anamount sufficient to cure or at least partially arrest the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective dose.” Amounts effective forthis use will depend on the disease condition being treated as well asby the judgment of the attending clinician depending upon factors suchas the severity of the inflammation, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient are in the form ofpharmaceutical compositions described above. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention willvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. For example, for intravenous administration, the dose willtypically be in the range of about 20 mg to about 500 mg per kilogrambody weight, preferably about 100 mg to about 300 mg per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.1 mg to 1 mg per kilogram body weight. Effective dosescan be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

EXAMPLES

In the discussion above and in the examples below, the followingabbreviations have the following meanings. If an abbreviation is notdefined, it has its generally accepted meaning.

7-methyl/MTL = 1-methylthio-7-deoxy-7- methyllincosamine apt = apparenttriplet atm = atmospheres Bn = benzyl Boc = tert-butoxycarbonylprotecting group br s = broad singlet BSTFA = N,O-bis(trimethylsilyl)trifluoroacetamide Cbz = carbonyloxybenzyloxyprotecting group CDCl₃ = deuterated chloroform CD₃OD = deuteratedmethanol cfu = colony forming units D = doublet DAST =dimethylaminosulfurtrifluoride dd = doublet of doublets dddd = doubletof doublets of doublet of doublets dt = doublet of triplets DCE =dicholoroethane DCM = dichloromethane DIEA = diisopropyethylamine DMAP =dimethylaminopyridine DMF = dimethylformamide DMSO = dimethyl sulfoxideDPPA = diphenylphosphoryl azide ED₅₀ = dose therapeutically effective in50% of the population Equiv = equivalents ESMS = electrospray massspectrometry Et = ethyl EtOAc = ethyl acetate Et₂O = diethyl ether g =grams h = hours HATU = O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HOBT = 1-hydroxybenzotriazolehydrate ¹H NMR = Hydrogen Nuclear Magnetic Resonance spectroscopy HPLC =high pressure liquid chromatography Hz = hertz IC₅₀ = concentration ofthe test compound which achieves a half-maximal inhibition of symptoms J= coupling constant in hertz L = liters LD₅₀ = dose lethal to 50% of thepopulation LiHMDS = lithium hexamethyldisilazide LiN(iPr)₂ = lithiumdiisopropylnitride m = multiplet M = molar MCPBA =2-(4-chloro-o-tolyloxy) acetic acid Me = methyl MeCN = acetonitrile MeOH= methanol mg = milligrams MHz = megahertz Min = minutes mL =milliliters Mm = millimeter mmol = millimol MS(ESPOS) = massspectrometry by positive mode electrospray ionization MS(ESNEG) = MassSpectrometry by negative mode electrospray ionization MTL =1-methylthiolincosamine (methyl 6- amino-6,8-dideoxy-1-thio-erythro-α-D-galacto-octopyranoside) N = normal NMR = nuclear magnetic resonance OBz= benzyloxy protecting group OtBu = tert-butoxy Pd/C = palladium/carbonpg = picograms Ph = phenyl Pro = L-proline psi = pounds per square inchq = quartet q.v. = quantitative R_(f) = Retention factor rt = roomtemperature s = singlet sat. = saturated t = triplet TEA = triethylamineTFA = trifluoroacetic acid THF = tetrahydrofuran TLC = thin layerchromatography μg = micrograms μL = microliters μm = micromolar v/v =volume by volume w/w = weight by weight

Additionally, the term “Aldrich” indicates that the compound or reagentused in the following procedures is commercially available from AldrichChemical Company, Inc., 1001 West Saint Paul Avenue, Milwaukee, Wis.53233 USA; the term “Fluka” indicates that the compound or reagent iscommercially available from Fluka Chemical Corp., 980 South 2nd Street,Ronkonkoma N.Y. 11779 USA; the term “Lancaster” indicates that thecompound or reagent is commercially available from Lancaster Synthesis,Inc., P.O. Box 100 Windham, N.H. 03087 USA; the term “Sigma” indicatesthat the compound or reagent is commercially available from Sigma, P.O.Box 14508, St. Louis Mo. 63178 USA; the term “Chemservice” indicatesthat the compound or reagent is commercially available from ChemserviceInc., Westchester, Pa., USA; the term “Bachem” indicates that thecompound or reagent is commercially available from Bachem BioscienceInc., 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia,Pa. 19406 USA; the term “Maybridge” indicates that the compound orreagent is commercially available from Maybridge Chemical Co.

Trevillett, Tintagel, Cornwall PL34 OHW United Kingdom; the term “RSP”indicates that the compound or reagent is commercially available fromRSP Amino Acid Analogs, Inc., 106 South St., Hopkinton, Mass. 01748,USA, and the term “TCI” indicates that the compound or reagent iscommercially available from TCI America, 9211 North Harborgate St.,Portland, Oreg., 97203, OR, USA; the term “Toronto” indicates that thecompound or reagent is commercially available from Toronto ResearchChemicals, Inc., 2 Brisbane Rd., New York, ON, Canada M3J2J8; the term“Alfa” indicates that the compound or reagent is commercially availablefrom Johnson Matthey Catalog Company, Inc. 30 Bond Street, Ward Hill,Mass. 018350747; and the term “Nova Biochem” indicates that the compoundor reagent is commercially available from Nova Biochem USA, 10933 NorthTorrey Pines Road, P.O. Box 12087, La Jolla Calif. 92039–2087.

In the examples below, all temperatures are in degrees Celsius (unlessotherwise indicated) and the following general procedures are used toprepared the compounds as indicated. It will be appreciated by one ofskill in the art that the following general procedures are meant to beillustrative only and that the methods may be broadened to synthesizeother compounds of the subject invention.

General Procedures

Method A

Methyl 6-amino-6,8-dideoxy-1-thio-erythro-α-D-galacto-octopyranoside 1a(R¹=Me) (MTL) was prepared as described by Hoeksema, H. et al., J. Am.Chem. Soc., 1967, 89, 2448–2452. N-(Benzyloxycarbonyloxy)succinimide(5.8 g 23.1 mmol) and 1a (5.0 g, 19.7 mmol) were suspended in pyridine(40 mL) and stirred under N₂ atmosphere 36 h. The reaction mixture wascooled to 0° C. and then bis-N,O-trifluoroacetamide (15.7 mL, 59.0 mmol)was added by syringe over 2 min. The reaction mixture was allowed towarm to rt and stirred for 42 h. Toluene (100 mL) was added and thereaction mixture was evaporated to dryness. The residue was taken up inethyl acetate (400 mL). The organic solution was washed quickly with 10%citric acid (200 mL), H₂O (3×100 mL), saturated NaHCO₃ (100 mL), andbrine (2×100 mL), and dried over Na₂SO₄ and evaporated to dryness.Chromatography of the crude product on silica 10% EtOAc/Hexanescontaining 0.2% TEA after co-evaporation from toluene (100 mL) andcyclohexane (2×100 mL) provided the protected product 1b (P=Cbz, R¹=Me)(7.2 g, 54%) as a colorless oil.

¹H NMR (300 MHz, CD₃SOCD₃) δ 7.34–7.31 (m, 5), 7.05 (d, J=8.2, 1), 5.19(d, J=5.8, 1), 5.01 (d, J=1.6, 2), 3.99 (apt dt, J=5.5, 9.3, 9.3, 2),3.93–3.86 (m, 3), 3.49 (dd, J=2.5, 9.6, 1), 2.01 (s, 3), 1.03 (d, J=6.3,3), 0.10 (s, 9), 0.09 (s, 9), 0.04 (m, 18).

To dimethylsulfoxide (413 μL, 5.82 mmol) in DCM (1.5 mL) cooled to −72°C. was added oxalyl chloride 2 M in DCM (1.49 mL, 2.98 mmol) over 1 min.After 25 min the protected product 1b (1.92 g, 2.84 mmol) in DCM (4.0mL) was added by cannula. The resulting reaction mixture was stirred for25 min and then allowed to warm to −50° C. (dry ice acetonitrile) andmaintained at this temperature for 2 h. To the reaction mixture wasadded TEA (1.29 mL, 3.30 mmol). After 25 min the reaction mixture wasdiluted with EtOAc (300 mL). The resulting organic solution was washedquickly with 5% citric acid (300 mL), H₂O (2×300 mL), saturated NaHCO₃(100 mL), brine (100 mL) dried over Na₂SO₄ and evaporated to drynesswith the aid of toluene (100 mL) to provide the product 1c. The product1c (P=Cbz, R¹=Me) was obtained as a colorless crystalline solid (1.60 g,94%) after co-evaporation with n-pentane and removal of residual solventunder high vacuum.

¹H NMR (300 MHz CDCl₃) δ 7.37–7.33 (m, 5), 5.60 (m, 1), 5.21 (d, J=5.2,1), 5.17 (d, J=12.4, 1), 5.08 (d, J=12.4, 1), 4.74 (m, 1), 4.16–4.12 (m,2), 3.87 (d, J=2.2, 1), 3.69 (dd, J=2.5, 9.3, 1), 2.01 (br s, 3), 1.90(s, 3), 0.19 (s, 9), 0.16 (s, 9), 0.15 (s, 9).

Method B

The Boc-protected product 1c (P=Boc, R¹=Me) may be prepared in generalas outlined above. 1a (R¹=Me) (MTL) (Dried at 50° C. high vacuum) (21.8g, 86 mmol) was suspended in methanol (200 mL) and TEA (26 mL) and wascooled to 0° C. on ice. To this mixture di-t-butyldicarbonate (57.0 g,0.26 mol) was added. The reaction mixture was then stirred overnight atrt. To the reaction mixture was added toluene (100 mL). The solventswere removed to a total volume of 100 mL, leaving a thick suspension towhich cyclohexane (300 mL) was added. The resulting solid precipitatewas triturated, then filtered and washed with cyclohexane, ether, andpentane and dried to constant weight. The crude Boc-protected productwas used without further purification (87%).

TLC R_(f)=0.75 (10% MeOH/DCM); MS(ESPOS): 354 (M+H); ¹H NMR (300 MHz,CD₃OD) δ 0.14 (d, J=6.3, 3), 1.43 (s, 9), 2.07 (s, 3), 3.55 (dd, J=3.3,10.43, 1), 3.84–4.08 (m, 3), 4.10–4.15 (m, 2), 5.25 (d, J=5.5, 1).

To N-Boc-1-methylthiolincosamide (240 mg, 0.68 mmol) in DMF (5 mL, BSTFA(0.52 mL, 2.0. mmol) and triethylamine (0.14 mL, 1.42 mmol) were addedat 0° C. and then stirred at rt overnight. DMF was removed and the crudeproduct was quickly passed through a silica gel column (pretreated with2% TEA in ethyl acetate) eluting with 10% ethyl acetate in hexanes 1b(P=Boc, R¹=Me) (350 mg, 95%). To oxalyl chloride (0.16 mL, 0.78 mmol) indichloromethane (5 mL) at −60° C., dimethylsulfoxide (0.22 mL, 0.78mmol) was added slowly and then stirred for 15 min. After which, 1b (370mg, 0.65 mmol) in DCM (5 mL) was added slowly. The reaction mixture wasstirred for 45 min, during which the reaction temperature was raised to−40° C. Triethylamine (0.70 mL, 3.25 mmol) was then added and thestirring continued for an additional 15 min at −40° C. It was thenextracted with DCM (100 mL) and washed with 10% citric acid (50 mL). Theresidue obtained on removal of solvent was then purified on silica gelcolumn using 10% ethyl acetate in hexanes as eluent 1c (P=Boc, R¹=Me) asa colorless oil (289 mg, 78%).

TLC: R_(f)=0.60 (10% EtOAc/Hexanes); MS(ESPOS): 590 (M+23); ¹H NMR (300MHz, CDCl₃) δ 0.11 (s, 18), 0.17 (s, 18), 1.40 (s, 9), 1.84 (s, 3), 2.26(s, 3), 3.63 (dd, J=2.7, 9.34, 1), 3.82 (d, J=1.9, 1), 4.01–4.12 (m, 2),5.15 (d, J=5.5, 1).

Method C

Triphenylphosphonium bromide (3.29 g, 9.2 mmol) and potassiumtert-butoxide (715 mg, 6.4 mmol) under N₂ atmosphere were suspended intoluene (31 mL) with vigorous stirring. After 4 h protected product 1c(P=Cbz, R¹=Me) (1.4 g, 2.36 mmol) in toluene (20 mL) was added bycannula. The resulting reaction mixture was stirred 2 h and then dilutedwith EtOAc (250 mL). The resulting organic solution was washed quicklywith H₂O (2×100 mL), brine (1×100 mL) dried over Na₂SO₄ and evaporatedto dryness. Chromatography of the crude producton silica 6%EtOAc/Hexanes containing 0.2% TEA gave the alkene product 2a (P=Cbz,R¹=Me, R^(2′)=CH₂) as a colorless oil that crystallized afterco-evaporation from toluene and cyclohexane (0.65 g, 46%).

1H NMR (300 MHz CDCl₃) δ 7.35–7.27 (m, 5), 6.36 (d, J=7.1, 1), 5.24 (d,J=5.5, 1), 5.08 (m, 4), 4.34 (m, 1), 4.16 (m, 2), 3.88 (d, J=2.2, 1),3.61 (dd, J=2.2, 9.3 1), 2.20 (s, 3), 1.79 (s, 3), 0.17–0.13 (m, 27).

The product 2a (P=Cbz, R^(1′)=Me, R²=CH2) (490 mg, 0.82 mmol) in ethanol(50 mL) was added to 10% palladium on carbon (Degussa wet form 50% w/wwater) (700 mg) in a par bottle. The bottle was purged, and charged withH₂ to 65 psi and shaken 24 h. The reaction mixture was filtered throughcelite, rinsed with methanol. The organic solution was transferred to aresin funnel containing dry, washed Dowex™ 50w-400x H⁺ form (0.8 g) andshaken for 10 min. After washing the resin with methanol three times andwater two times, the saturated product 2b (R¹=Me, R²=Me) was eluted fromthe resin by washing with 5% TEA in MeOH (35 mL, ×10 min×5). Thecombined filtrate was evaporated to dryness, co-evaporated from EtOHtwice and lyophilized from 1:1 MeCN/H₂O to give the product as acolorless powder (198.4 mg 96%).

¹H NMR (300 MHz, D₂O) δ 5.17 (d, J=5.8, 1), 3.97–3.84 (m, 3), 3.52 (dd,J=3.0, 10.0, 1), 2.82 (dd, J=4.4, 8.5, 1), 1.94 (s, 3), 1.89–1.81 (m,1), 0.82 (d, J=6.9, 3), 0.72 (d, J=6.9, 3). MS(ESPOS): 252.2 [M+H],(ESNEG): 250.4 [M−H].

Method D

In the alternative when a Boc-protecting group is used,methyltriphenylphosphonium bromide (12 g, 33.6 mmol) and potassiumt-butoxide (3 g, 26.7 mmol) were taken in THF (70 mL) at 0° C., andstirred at rt for 4 h. Then Boc-protected product 1c (P=Boc, R¹=Me) (4.7g, 8.2 mmol) in THF (30 mL) was added and stirred at rt for 2 h. Afterwhich it was extracted with EtOAc (300 mL), washed with brine (100 mL)and dried over sodium sulfate. The crude alkene product 2a (P=Boc,R¹=Me, R^(2′)=CH₂) was purified on silica gel column chromatographyusing 10% EtOAc in Hexane as eluent (4.1 g, 87.6%).

TLC: R_(f)=0.5 (10% of EtOAc in Hexane): ¹H NMR (300 MHz, CD₃OD) δ 7.24(m, 2), 5.22 (d, J=5.7, 1), 4.21 (m, 1), 4.09 (m, 2), 3.87 (d, J=2.4,1), 3.60 (dd, J=2.7, 9.3, 1), 1.99 (s, 3), 1.76 (s, 3); 1.43 (s, 9);MS(ESPOS): 444 (M−2TMS+Na).

To the product 2a (P=Boc, R¹=Me, R^(2′)=CH₂) in methanol (30 mL), Dowex™H⁺ resin (1 g) was added and stirred at rt for 1 h. The resin wasfiltered and the product obtained on removal of solvent (2.4 g, 6.8mmol,) was taken in MeOH (30 mL). Pd/C (2.5 g) was added andhydrogenated at 55 psi overnight. The crude product obtained onfiltering and removal of solvent was purified on silica gel columnchromatography using 10% MeOH in DCM to provide Boc-protected 7-MethylMTL as a white solid (2.06 g, 86%). TLC R_(f)=0.5 (10% of MeOH in DCM).

¹H NMR (300 MHz, CD₃OD) δ 5.23 (d, J=5.4, 1), 4.11 (m, 1), 3.97 (d,J=10.2, 1), 3.84 (m, 1), 3.52 (m, 1), 2.08 (s, 3), 1.44 (s, 9), 1.14 (m,1), 0.93 (d, J=6.9, 3), 0.85 (d, J=6.9, 3); MS(ESPOS): 351(M+H).

To the Boc-protected 7-Methyl MTL (150 mg, 0.43 mmol) in dichloroethane(6 mL), dimethylsulfide (0.16 mL, 2.5 mmol) was added, followed by TFA(2 mL), water (0.16 mL) and stirred at rt for 1 h. The solvent wasremoved to obtain the crude product 2b (R¹=Me, R²=Me). Afterpurification on silica gel column chromatography using 30% MeOH in DCMas eluent, the product 2b (R¹=Me, R²=Me) was obtained identical in allrespects to the material obtained from method C.

Method E

Sodium hydride (80 mg, 3.3 mmol) under N₂ atmosphere was suspended inTHF (4 mL) with vigorous stirring. The suspension was cooled to −30° C.and diethyl(cyanomethyl)phosphonate (805 μL, 5.0 mmol) was added. After30 min protected product 1c (P=Cbz, R¹=Me) (1.0 g, 1.7 mmol) in THF (3mL) was added by cannula. The resulting reaction mixture was stirred 4 hand then diluted with EtOAc (250 mL). The resulting organic solution waswashed quickly saturated aqueous NaHCO₃ (1×100 mL), brine (1×50 mL)dried over Na₂SO₄ and evaporated to dryness. Chromatography of the crudeproduct on silica 6% EtOAc/Hexanes to 10% EtOAc/Hexanes containing 0.2%TEA gave the protected alkene product 2a (P=Cbz, R¹=Me, R^(2′)=CHCN) asa colorless oil (0.38 g, 37%). MS(ESPOS): 625.5.2 [M+H], ES(NEG): 659.5[M+Cl].

The product 2a (P=Cbz, R¹=Me, R^(2′)=CHCN) (180 mg, 0.29 mmol) inethanol (15 mL) was added to 10% palladium on carbon (Degussa wet form50% w/w water) (300 mg) in a Parr bottle and concentrated HCl (29 μL)was added. The bottle was purged, and charged with H₂ to 65 psi andshaken for 24 h. The reaction mixture was filtered through celite,rinsed with methanol. The organic solution was transferred to a resinfunnel containing dry, washed Dowex™ 50w-400x H⁺ form (1 g) and shaken10 min. After washing the resin with methanol twice and water, thesaturated product 2b (R¹=Me, R²=CH₂CN) was eluted from the resin bywashing with 5% TEA in MeOH (20 mL×20 min×3) and MeCN (20 mL×20 min).The combined organic filtrate was evaporated to dryness lyophilized from1:1 MeCN/H₂O to give the product 2b (R¹=Me, R²=CH₂CN) as a colorlesssolid (70 mg, 91%). ES(NEG): 275.3 [M−H].

Method F

To the protected product 1c (P=Cbz, R¹=Me) (0.75 g, 1.3 mmol) in THF(7.3 mL) was added MeMgCl (3M) in THF (7.0 mL 2.1 mmol) at 0° C. Over 30min the reaction mixture was warmed to 4° C. and after 4 h the reactionmixture was quenched with 1:3 saturated aqueous NH₄Cl/H₂O (10 mL). Thequenched mixture was diluted to 100 mL with water and extracted with DCM(4×50 mL). The combined organic phase was dried and evaporated. Theresidue was dissolved in 1:2:4 H₂O/HOAc/THF (100 mL) and stirred for 20h, and then evaporated with the aid of toluene (2×100 mL).Chromatography 10:1 to 10:2 DCM/MEOH gave product 3a (P=Cbz, R¹=Me,R²=Me) (153 mg, 31%).

(ESNEG): 399.5 [M−H].

3a (P=Cbz, R¹=Me, R²=Me) (79 mg, 0.2 mmol) in ethanol (10 mL) was addedto 10% palladium on carbon (Degussa wet form 50% w/w water) (400 mg) ina Parr bottle. The bottle was purged, and charged with H₂ to 65 psi andshaken 6 h. The reaction mixture was filtered through celite, rinsedwith methanol. The combined filtrate was evaporated to dryness andlyophilized from 1:1 MeCN/H₂O to give the product 3b (R¹=Me, R²=Me) as acolorless powder (42 mg, 80%).

¹H NMR (300 MHz, D₂O) δ 5.33 (d, J=5.8, −1), 4.83–4.06 (m, 3), 3.65–3.60(m, 1), 3.06–3.03 (m, 1), 2.18 (s, 3), 1.30 (s, 3), 1.23 (s, 3).MS(ESPOS): 268.4 [M+H], MS(ESNEG): 266.2 [M−H].

Method G

To the Boc-protected product 1c (P=Boc, R¹=Me) (100 mg, 0.18 mmol) inmethanol (3 mL), O-trimethylsilylhydroxylamine (0.10 mL, 0.88 mmol) wasadded and stirred at rt overnight. The solvent was removed to obtain thecrude Boc-protected product 4a (P=Boc, R¹=Me, R⁷=H). To the crudeproduct 4a (95 mg, 0.15 mmol), 30% trifluoroacetic acid indichloroethane (10 mL) and dimethyl sulfide (0.5 mL) were added andstirred for 1 h. The solvent was removed and the product 4b (R¹=Me,R⁷=H) was taken as such for the next step.

TLC: R_(f)=0.35 (10% MeOH/DCM); MS(ESPOS): 267 (M+H); ¹H NMR (300 MHz,CD₃OD) δ 1.96 (s, 3), 2.09 (s, 3), 3.58 (dd, J=3.3, 10.2, 1), 3.90 (s,1), 4.11 (dd, J=5.7, 10.20, 1), 4.19(d, J=5.4, 1), 4.50 (d, J=5.1, 1),5.36 (d, J=5.7, 1).

Method H

To the Boc-protected product 1c (P=Boc, R¹=Me) (100 mg, 0.176 mmol) inmethanol (4 mL) and water (1 mL), O-alkylhydroxylamine hydrochloride(for example, O-methylhydroxylamine hydrochloride) (60 mg, 0.70 mmol)and sodium acetate (57 mg, 0.70 mmol) were added and heated at 80° C.for 3 h and then stirred at rt overnight. The solvent was removed underhigh vacuum to obtain the crude Boc-protected product 4a (P=Boc, R¹=H,R⁷=Me). The crude product 4a was taken in 30% trifluoroacetic acid indichloroethane (10 mL), dimethylsulfide (0.5 mL) and stirred for 1 h atrt. The solvent was removed and the residue was kept under high vacuumfor 1 h and the product 4b (R¹=Me, R⁷=Me) was taken as such for the nextstep.

TLC: R_(f)=0.63 (10% MeOH/DCM); MS(ESPOS): 281 (M+H). ¹H NMR (300 MHz,CD₃OD) δ 1.95 (s, 3), 2.08 (s, 3), 3.60 (dd, J=3.3, 10.20, 1), 3.92 (s,3), 4.13 (dd, J=4.8, 10.20, 1), 4.49 (d, J=1.2, 1), 5.38 (d, J=5.4, 1).

Method I

To the Boc-protected product 1c (P=Boc, R¹=Me) (500 mg, 0.88 mmol) inTHF (10 mL), tetrabutylammonium fluoride (2.5 mmol, 1 M in THF) wasadded and the reaction mixture was stirred at rt for 1 h. The solventwas removed and the residue was purified on silica gel column using 5%methanol in dichloromethane as eluent. The product (111 mg, 0.31 mmol)obtained from the column was then taken in a mixture of dichloromethane(3 mL) and pyridine (3 mL) to which acetic anhydride (0.5 mL, 10.6 mmol)and dimethylaminopyridine (80 mg, 1.7 mmol) were added and stirred at rtovernight. The solvent was removed and the crude product was purified onsilica gel column using 30% ethyl acetate in hexanes as eluent toprovide 5a (P=Boc, R¹=Me) (58 mg, 38%).

TLC: R_(f)=0.73 (50% EtOAc/Hexanes); MS(ESPOS): 500 (M⁺Na). ¹H NMR (300MHz, CDCl₃) δ 1.38 (s, 9), 1.91 (s, 3), 1.98 (s, 3), 2.07 (s, 3), 2.18(s, 3), 4.33 (m, 1), 4.72 (m, 1), 4.94 (m, 1), 5.21 (m, 2), 5.45 (s, 1),5.57 (m, 1).

To product 5a (P=Boc, R¹=Me) (158 mg, 0.331 mmol) in DCM (5 mL),dimethylaminosulfurtrifluoride (732 μL, 3.31 mmol) was added and stirredovernight. More DCM was added and the organic portion was washed withsodium bicarbonate. The residue obtained on removal of solvent waspurified on silica gel column chromatography using 20% ethyl acetate inhexanes as eluent (100 mg, 60%) to provide the protected product (P=Boc,R¹=Me). The Boc-protected product was taken up in 30% trifluoroaceticacid in dichloroethane and dimethylsulfide and stirred for 1 h at rt.The solvent was removed to provide the product 5b (R¹=Me).

TLC: R_(f)=0.63 (40% MeOH/Hexanes); MS(ESPOS): 522 (M+23). ¹H NMR (300MHz, CDCl₃) δ 1.40 (s, 9), 1.69 (t, J=18.9, 3), 1.98 (s, 3), 2.08 (s,6), 2.13 (s, 3), 4.22–4.30 (m, 1), 4.53 (dd, J=10.9, 25.3, 1), 5.16–5.28(m, 2), 5.52 (s, 1), 5.63 (d, J=5.2, 1).

Method J

Enolization (LiHMDS) and alkylation of 7a with4-bromo-2-methyl-2-butenyl afforded a mixture of diastereomers of thelactam 7b (R^(9′)=2-methyl-2-butenyl) (61%) according to the literatureprocedure by Zhang, R.; et al., Journal of the American ChemicalSociety. 1998, 120, 3894–3902. Compound 7a is commercially availablefrom vendors such as Bachem. Alternatively, 7a can be prepared bymethods well known in the art for an example see Baldwin, et al.;Tetrahedron, 1989, 45, 7449–7468.

The lactam 7b was reduced to the pyrrolidine 7c(R^(9′)=2-methyl-2-butenyl) (70%) by the two-step sequence involvingsuperhydride reduction of the lactam to the hemiaminal and thesubsequent reduction of the hemiaminal with Et₃SiH/BF₃.OEt₂. Thepyrrolidine 7c (R^(9′)=2-methyl-2-butenyl) (778 mg, 2.08 mmol), 10%palladium on carbon (230 mg), in anhydrous methanol (25 mL) wassubjected to Parr hydrogenolysis at 50 psi for 5 h. The reaction mixturewas filtered through a celite pad and washed several times withmethanol. The combined washings and filtrate were evaporated to dryness,affording, without further purification, a colorless oil 7d(R⁹=2-methylbutyl).

TLC: R_(f)=0.3 [Solvent system: DCM:hexanes:MeOH(6:5:1)]. MS(ESNEG):284.5 [M−H]₌.

Method K

Enolization (LiHMDS, 33 mmol, 33 mL, 1.1 equiv) and alkylation of 7a(9.47 g, 29.7 mmol, 1 equiv) with cis-1-bromo-2-pentene (4.21 mL, 35.6mmol, 1.2 equiv), in anhydrous THF at −78° C. under nitrogen, afforded amixture of diastereomers of the lactam 7b (R^(9′)=2-pentenyl) (43.2%)after silica gel purification. The lactam 7b (3.96 g, 10.22 mmol) wasreduced to the pyrrolidine 7c (R^(9′)=2-pentenyl) by the two-stepsequence involving superhydride reduction of the lactam to thehemiaminal, at −78° C. in anhydrous THF, and the subsequent reduction ofthe hemiaminal with Et₃SiH/BF₃OEt₂ in anhydrous DCM at −78° C. affording7c (R⁹′=2-pentenyl) (71%) after silica gel purification. The pyrrolidine7c (2.71 g, 7.26 mmol), 10% palladium on carbon (560 mg), in anhydrousmethanol (30 mL) was subjected to Parr hydrogenolysis at 50 psi for 5 h.The reaction mixture was filtered through a celite pad and washedseveral times with methanol. The combined washings and filtrate wereevaporated to dryness, affording, without further purification, acolorless oil 7d (R⁹=pentyl) (1.68 g, 80%).

TLC: R_(f)=0.3 [Solvent system:DCM:hexanes:MeOH(6:5:1)]. MS(ESNEG):284.5 [M−H].

Method L

Ozonolysis of 7d (R⁹=2-methylbutyl) in anhydrous dichloromethanefollowed by treatment with DMS at −78° C. afforded aldehyde 8a (77%).4-Fluorobenzyl phosphonium chloride (0.87 g, 2.13 mmol) and potassiumt-butoxide (0.17 g, 1.48 mmol) were suspended in toluene under nitrogenwith vigorous stirring. After 4 h, a solution of aldehyde 8a (204 mg,0.59 mmol) in toluene (4.6 mL) was added drop-wise. The reaction mixturewas stirred at rt for 2 h and diluted with ethyl acetate (50 mL). Theorganic layer was washed with water (2×20 mL), brine, dried andconcentrated. The residue was purified by chromatography to give a clearsyrup 8b (R^(9′)=3-(4-fluorophenyl)prop-2-enyl) (171 mg).

To a solution of 8b (R^(9′)=3-(4-fluorophenyl)prop-2-enyl) (171 mg, 0.39mmol) in MeOH (25 mL) in a Parr bottle was added 10% palladium on carbon(Degussa wet form 50% w/w water) (200 mg). The bottle was purged andcharged with H₂ to 40 psi, and shaken for 4 h. The reaction mixture wasfiltered through celite and rinsed with MeOH. The filtrate wasconcentrated to give a yellow oil 8c (R⁹=3-(4-fluorophenyl)propyl) (120mg).

MS(ESPOS): 374.5 [M+Na]⁺, MS(ESNEG): 350.3 [M−H]⁻.

Method M

4-Chlorobenzyl phosphonium chloride (0.95 g, 2.24 mmol, 3.9 equiv) andpotassium t-butoxide (0.17 g, 1.55 mmol, 2.7 equiv) were suspended intoluene (7.5 mL) under nitrogen with vigorous stirring. After 4 h, asolution of aldehyde 8a (200 mg, 0.58 mmol, 1 equiv) in toluene (4.9 mL)was added dropwise. The reaction mixture was stirred at rt for 2 h anddiluted with ethyl acetate (50 mL). The organic layer was washed withwater (2×20 mL), brine, dried and concentrated. The residue was purifiedby chromatography to give a clear syrup 8b(R^(9′)=3-(4-chlorophenyl)prop-2-enyl) (216 mg, 82%).

MS(ESPOS): 478.5 [M+Na]⁺, MS(ESNEG): 454.4 [M−H]⁻.

To a solution of 8b (R^(9′)=3-(4-chlorophenyl)prop-2-enyl) (147 mg, 0.32mmol) in cyclohexane (50 mL) was added 10% palladium on carbon (Degussawet form 50% w/w water) (86 mg). The reaction mixture was stirred at rtunder 1 atm H₂ overnight. The reaction mixture was filtered throughcelite and rinsed with MeOH. The filtrate was concentrated to give thealkane product 8c (R⁹=3-(4-chlorophenyl)propyl) as a clear oil (131 mg,89%). To a solution of the alkane (131 mg, 0.29 mmol, 1 equiv) in THF (3mL) and water (1 mL) was added lithium hydroxide monohydrate (60 mg,1.43 mmol, 5 equiv). The reaction mixture was stirred at rt overnight.The THF was removed under vacuum. The residue was diluted with water (5mL) and washed with ether (10 mL). The aqueous layer was taken up inethyl acetate (60 mL) and partitioned with 10% citric acid (30 mL). Theorganic layer was washed with water and brine, dried and concentrated togive a clear syrup 8c (R⁹=3-(4-chlorophenyl)propyl) (105 mg, 100%).

MS(ESPOS): 390.4 [M+Na]⁺, 268.4 [M−Boc+H]⁺.

Method N

To a solution of aldehyde 8a (406.5 mg, 1.17 mmol, 1 equiv) in dimethylacetamide (0.25 mL) at 0° C. was added dibromodifluoromethane (0.21 mL,2.34 mmol, 2 equiv). To the stirred mixture was added a solution oftriphenylphosphine (0.61 g, 2.34 mmol, 2 equiv) in dimethyl acetamide(0.5 mL) over a period of 20 minutes under nitrogen. The reactionmixture was warmed to rt and stirred for 30 minutes, and then was addedto an activated zinc (0.25 g, 3.82 mmol, 3.3 equiv) with the aid ofdimethyl acetamide (0.3 mL). The resulting reaction mixture was stirredat 110° C. for 1 h and cooled to rt and filtered with the aid ofdimethylacetamide (7 mL). The filtrate was poured into ice water (100mL) and extracted with ether (150 mL). The ether layer was washed withbrine, dried and concentrated. The residue was purified bychromatography to give a clear oil 8b (R^(9′)=3,3-difluoroprop-2-enyl)(182 mg, 41%).

MS(ESPOS): 282.4 [M−Boc+H]⁺.

To a solution of 8b (R^(9′)=3,3-difluoroprop-2-enyl) (126 mg, 0.33 mmol)in MeOH (35 mL) was added 10% palladium on carbon (Degussa wet form 50%w/w water) (120 mg). The reaction mixture was stirred at rt underhydrogen (1 atm) overnight and was filtered through celite with the aidof MeOH. The filtrate was concentrated to give a clear syrup 8c(R⁹=3,3-difluoropropyl) (97 mg, 100%).

MS(ESPOS): 194.4 [M−Boc+H]⁺, MS(ESNEG): 292.4 [M−H]⁻.

Method O

To a solution of aldehyde 8a (258 mg, 0.74 mmol, 1 equiv) in THF (3 mL)at 0° C. was added tetraallyltin (178 μL, 0.74 mmol, 1 equiv), followedby the drop-wise addition of boron trifluoride etherate (94.3 μL, 0.74mmol, 1 equiv) over a period of 15 min. The reaction mixture was stirredat 0° C. for 1.5 h. Then a solution of potassium fluoride (125 mg) inwater (1.25 mL) was added. The resulting mixture was warmed to rt andstirred at rt for 20 min. This was followed by the addition of methanol(10 mL) and the resulting mixture was stirred at rt for another 20 min.The reaction mixture was filtered over celite. The filtrate wasevaporated to dryness. The residue was diluted with dichloromethane (100mL), washed with water (50 mL), dried, concentrated and purified bychromatography to give a clear oil 9a (R⁹=2-hydroxypent-4-enyl) (261 mg,90%): MS(ESPOS): 412.5 [M+NA]⁺, 290.4 [M−Boc+H]⁺.

To a solution of dimethylsulfoxide (0.17 mL, 2.42 mmol, 3 equiv) indichloromethane (0.5 mL) at −72° C. was added a 2 M solution of oxalylchloride in dichloromethane (0.61 mL, 1.21 mmol, 1.5 equiv) over aperiod of 1 min. The mixture was stirred at −72° C. for 25 min, followedby the drop-wise addition of a solution of the alcohol 9a (314 mg, 0.81mmol, 1 equiv) in dichloromethane (1.4 mL) over a period of 2 min. Thereaction mixture was stirred at −72° C. for 25 min, then warmed to −50°C. and stirred for an additional 2 h. Triethylamine (0.56 mL, 4.04 mmol,5 equiv) was added and stirred at −50° C. for 25 min. The mixture wasdiluted with ethyl acetate (100 mL), washed with 5% citric acid (100mL), water, saturated aqueous NaHCO₃ and brine, dried, evaporated andcoevaporated with anhydrous toluene to give a clear syrup 9b(R⁹=2-propenylcarboxymethyl) (287 mg, 92%). MS(ESPOS): 288.5 [M−Boc+H]⁺;MS(ESNEG): 386.2 [M−H]⁻.

To a solution of ketone 9b (225.1 mg, 0.58 mmol, 1 equiv) indichloromethane (2 mL) at −78° C. was added diethylaminosulfurtrifluoride (0.46 mL, 3.49 mL, 6 equiv). The reaction mixture was warmedto rt and stirred at rt for 3 h, followed by an addition of additional(diethylamino)sulfur trifluoride (0.46 mL, 3.49 mL, 6 equiv) at −78° C.The mixture was warmed to rt and stirred overnight. Then the mixture wasdiluted with dichloromethane (60 mL), washed with sat. aqueous NaHCO₃(1×), brine (1×), dried, and evaporated. The residue was purified bychromatography to give a yellow oil 9c (X, X=fluoro, flouro) (75 mg,32%).

MS(ESPOS): 310.5 [M−Boc+H]⁺.

To a solution of 9c (R⁹=2,2-difluoropent-4-enyl) (85 mg, 0.21 mmol) inMeOH (20 mL) was added 10% palladium on carbon (Degussa wet form 50% w/wwater) (100 mg). The reaction mixture was stirred at rt under hydrogen(1 atm) overnight, was filtered through celite with the aid of MeOH (10mL). To the filtrate was added 10% palladium on carbon (Degussa wet form50% w/w water) (130 mg). The reaction mixture was stirred at rt underhydrogen (1 atm) overnight, was filtered through celite with the aid ofMeOH (10 mL). The filtrate was concentrated to give haloalkylN-Boc-amino acid 9d (X,X=fluoro, fluoro) (67.7 mg, 100%) as a clearsyrup.

MS(ESPOS): 344.4 [M+Na]⁺, 222.4 [M−Boc+H]⁺ MS(ESNEG): 320.2 [M−H]⁻.

Method P

To 4-propylpyridine (2.5 g, 20 mmol), 30% hydrogen peroxide (2.4 g) wasadded and refluxed overnight. The solvent was removed and the resultingresidue was taken in DCM (30 mL). Trimethylsilyl cyanide (2.6 g, 26mmol) was added to the above solution followed by dimethylcarbamylchloride (2.8 g, 26 mmol), and the reaction mixture was stirred at rtovernight. Potassium carbonate (10%, 100 mL) was added. The organiclayer was separated, dried over sodium sulfate and then concentrated toobtain 4-propyl-2-cyanopyridine (2.5 g, 93%). The crude nitrile wasdissolved in aqueous hydrochloric acid (6N, 60 mL) and refluxedovernight. The 4-propyl-2-carboxylic acid pyridine 10b (R⁹=propyl) wasobtained after crystallization from acetonitrile (2 g, 71%).

MS(ESPOS): 166 (M+H); ¹H NMR (300 MH₂, CD₃OD) δ 8.75 (dd, J=9.0, 3.0,1), 8.42 (s, 1), 8.08 (dd, J=9.0, 3.0, 1), 3.00 (t, J=7.5, 2), 1.82 (m,2), 1.05 (t, J=7.2, 3).

A mixture of picolinic acid (Aldrich) (20 g, 162 mmol, 1 equiv) andsodium bromide (33.43 g, 325 mmol, 2 equiv) in thionyl chloride (81 mL)was refluxed for 5 h. The solvent was removed under vacuum. Absolutemethanol (160 mL) was added and the mixture was stirred at rt for 30minutes. The solvent was evaporated, and the residue was taken up in 5%sodium bicarbonate and extracted with ethyl acetate (3×). The organiclayers were combined and dried over MgSO₄ and evaporated. The residuewas purified by chromatography to give 4-chloropicolinic acid methylester as a white solid (19.9. g, 72%): ¹H NMR (300 MHz, CDCl₃) δ 8.63(d, J=5.4, 1), 8.13 (d, J=2.1, 1), 7.48 (dd, J=2.0, 5.3, 1), 4.00 (s,3).

A mixture of 4-chloropicolinic acid methyl ester (2.4 g, 14.1 mmol), 57%hydroiodic acid (13.3 mL) and 50% aqueous hypophosphorous acid (0.66 mL)was stirred at 85° C. for 2 h and then was stirred at 107° C. overnight.The mixture was cooled to 95° C. At this temperature over 30 minutes 10M sodium hydroxide aqueous solution (4.2 mL) was added, followed by theaddition of water (15.2 mL). The mixture was cooled to rt and stirred atrt for 1 h. The precipitate was filtered, washed with cold water anddried under high vacuum overnight to give 4-iodopipecolinic acid 13a(3.5 g, 66%): ¹H NMR (300 MHz, DMSO d₆) δ 8.39 (d, J=5.1, 1), 8.35 (d,J=1.8, 1), 8.07 (dd, J=1.7, 5.2, 1); MS (ESPOS): 250.2 [M+H]⁺.

To a mixture of 7-Me MTL HCl salt 2b (R¹=Me, R²=Me) (200 mg, 0.69 mmol,1 equiv) in dry DMF (1.8 mL) at 0° C. was added triethylamine (0.50 mL,3.61 mmol, 5.2 equiv), followed by the addition of BSTFA (0.28 mL, 1.04mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for 10minutes, and then was stirred at rt for 50 minutes. To the reactionmixture was added the acid 13a (341 mg, 0.90 mmol, 1.3 equiv) and HATU(423 mg, 1.11 mmol, 1.6 equiv). The reaction mixture was stirred at rtfor 3 h. The reaction mixture was evaporated to dryness, taken up inethyl acetate, washed with water (1×), sat. NaHCO₃ (1×) and brine. Theorganic layer was dried over Na₂SO₄ and evaporated to give a yellowresidue which was dissolved in methanol (20 mL) to which was added dryDowex™ resin (250 mg). The reaction mixture was stirred at rt for 1 h.The resin was removed by filtration and the crude product eluted with 2Mammonia in methanol. The methanolic eluent was evaporated, and theresulting residue was purified by chromatography to provide a whitesolid 13b (R¹=Me, R²=Me, R³=H) (250 mg, 75%): ¹H NMR (300 MHz, CD₃OD) δ8.46 (d, J=1.8, 1), 8.30 (d, J=5.4, 1), 7.98 (dd, J=1.8, 5.1, 1), 5.25(d, J=6.0, 1), 4.32–4.23(m, 2), 4.09 (dd, J=5.7, 10.2, 1), 3.87 (d,J=3.0, 1), 3.54 (dd, J=3.3, 10.2, 1), 2.24–2.15 (m, 1), 2.11 (s, 3),0.99–0.96 (m, 6); MS (ESPOS): 483.5 [M+H]⁺; MS (ESNEG): 481.4 [M−H]⁻

To a dry flask was added 13b (R1=Me, R2=Me, R3=H) (133.9 mg, 0.28 mmol,1 equiv), triphenylphosphine (46.7 mg, 0.18 mmol, 0.64 equiv), copper(I) iodide (33.9 mg, 0.18 mmol, 0.64 equiv), palladium acetate (20 mg,0.09 mmol, 0.32 equiv) and triethylamine (1.6 mL). The mixture wasdeaerated with nitrogen, followed by addition of3-prop-2-ynyl-cyclopentane (120 mg, 1.11 mmol, 4 equiv). The mixture wasstirred at 50 oC overnight. The solvent was removed under vacuum to givea dark residue. The residue was purified by chromatography to give 13c(R1=Me, R9′=3-cyclopentyl-prop-1-ynyl, R2=Me, R3=H) as a yellow solid(106 mg, 83%): 1H NMR (300 MHz, CD₃OD) δ 8.55 (d, J=4.8, 1), 7.98 (s,1), 7.47 (dd, J=1.7, 5.0, 1), 5.26 (d, J=5.4, 1), 4.33–4.22 (m, 2), 4.10(dd, J=5.5, 10.4, 1), 3.86 (d, J=3.3, 1), 3.55 (dd, J=3.3, 10.5, 1),2.49 (d, J=6.9, 2), 2.26–2.12 (m, 2), 2.11 (s, 3), 1.93–1.82 (m, 2),1.73–1.55 (m, 4), 1.43–1.31 (m, 2), 1.00–0.96 (m, 6); MS (ESPOS): 463.6[M+H]⁺; MS (ESNEG): 461.5 [M−H]⁻.

Method R

To a solution of 13a prepared in general method Q (5 g, 13.26 mmol) inmethanol (500 mL) was added a few drops of conc. sulfuric acid. Thereaction mixture was refluxed overnight. The solvent was evaporated andthe residue was purified by chromatography to give 4-iodopipecolinicacid methyl ester 14a as a yellow solid (3.0 g, 86%): ¹H NMR (300 MHz,CDCl₃) δ 8.49 (d, J=1.5, 1), 8.37 (d, J=5.4, 1), 7.85 (dd, J=1.6, 5.2,1), 4.00 (s, 3); MS (ESPOS): 264.3 [M+H]⁺.

To a dry flask were added 14a (1 g, 3.8 mmol, 1 equiv),triphenylphosphine (79.7 mg, 0.3 mmol, 0.08 equiv), copper (I) iodide(57.9 mg, 0.3 mmol, 0.08 equiv), palladium acetate (34.1 mg, 0.15 mmol,0.04 equiv) and triethylamine (14 mL). The mixture was deaerated withnitrogen, followed by addition of 3-butyn-1-ol (0.53 g, 7.6 mmol, 2equiv). The mixture was stirred at rt for 3 h. The solvent was removedunder vacuum to give a dark residue. The residue was purified bychromatography to give 14b (R^(9′)=3-hydoxy-but-1-ynyl) as a yellow oil(0.78 g, 100%): ¹H NMR (300 MHz, CDCl₃) δ 8.66–8.63 (m, 1), 8.09–8.08(m, 1), 7.43–7.40 (m, 1), 3.99 (s, 3), 3.88–3.82 (m, 2), 2.72 (t, J=6.3,2). MS (ESPOS): 206.4 [M+H]⁺.

To a solution of the above 14b (R^(9′)=3-hydoxy-but-1-ynyl) (0.78 g, 3.8mmol) in methanol (40 mL) was added 10% palladium on carbon (0.4 g). Theflask containing the reaction mixture was purged and charged withhydrogen (1 atm) and stirred at rt overnight. The palladium was removedby filtration and the filtrate was concentrated to give 14c(R⁹=3-hydroxybutyl) as an oil (0.77 g, 97%): ¹H NMR (300 MHz, CDCl₃) δ8.60 (d, J=4.5,1), 7.97 (d, J=1.2, 1), 7.29 (dd, J=1.6, 5.0, 1), 3.99(s, 3), 3.67 (t, J=6.3, 2), 2.72 (t, J=7.7, 2), 1.81–1.69 (m, 2),1.62–1.54 (m, 2); MS (ESPOS): 210.4 [M+H]⁺.

To 4-hydroxypyridine-2-carboxylic acid (200 mg, 1.4 mmol) in DMF (2 mL),potassium carbonate (397 mg, 2.8 mmol) was added followed byn-bromobutane (197 mg, 1.4 mmol), warmed at 60° C. for overnight. Thesolvent was removed to obtain the crude ester product. The crude ester(360 mg, 1.4 mmol) was dissolved in THF (4 mL), lithium hydroxide (72mg, 1.7 mmol) was added, and the reaction mixture stirred at roomtemperature for 2 h. The residue obtained on removal of solvent waspurified by silica gel chromatography using 10% MeOH in DCM to provide4-butoxypyridine-2-carboxylic acid 15a (R¹⁰=butyl) (100 mg, 43%). ¹H NMR(300 MHz, CD₃OD) δ 8.37 (d, J=6.0, 1), 7.63 (d, J=2.7, 1), 7.07 (dd,J=2.7, 6.0, 1), 4.15 (t, J=6.6, 2), 1.82 (m, 2), 1.54 (m, 2), 1.01 (t,J=7.5, 3). MS (ES⁻): 194 (M−1).

To 4-butoxypyridine-2-carboxylic acid 15a (R¹⁰=Butyl) (100 mg, 0.5 mmol)in DMF (2 mL), 7-methyl α-thiolincosaminide 2b (R¹=Me, R²=Me) (147 mg,0.5 mmol) was added, followed by HBTU (214 mg, 0.55 mmol) and DIEA (132mg, 1 mmol). The reaction mixture was stirred at rt for 2 h. Thensolvent was removed. Purification of the crude material was carried outby silica gel column chromatography to obtain compound 15b (R¹=Me,R²=Me, R³=H, R¹⁰=butyl) (201 mg, 91%):

¹H NMR (300 MHz, CD₃OD) δ 8.42 (m, 1), 7.96 (s, 1), 7.09 (m, 1), 5.27(d, J=5.4, 1), 4.10–4.87 (m, 3), 3.85 (d, J=3.3, 1), 3.76 (m, 1), 2.11(m, 4), 1.81 (m, 2), 1.49 (m, 4), 0.99 (m, 9). MS (METHOD ES+): 428(M+1).

To a solution of the pyridine 15b (R¹=Me, R²=Me, R³=H, R¹⁰=butyl) (200mg, 0.46 mmol) in water (10 mL), AcOH (3 mL) and MeOH (2 mL), was addedPtO₂ (200 mg) and the resulting reaction mixture shaken under 55 psihydrogen overnight. Residual catalyst was removed by filtration throughcelite, and the solvent was removed to obtain the crude product.Purification was carried out by silica gel column chromatography using20% MeOH in DCM to obtain lincosamide analog 1 (R¹=Me, R²=Me, R³=H,R¹⁰=butyl) (12 mg, 6%). ¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.4, 1),4.22 (dd, J=10.2; 3.3, 1), 4.08 (m, 2), 3.81 (d, J=3.0, 1), 3.70 (m, 1),3.54 (m, 4), 3.43 (m, 2), 2.90 (m, 1), 2.41 (m, 1), 2.19 (m, 1), 2.10(s, 3), 1.45 (m, 6), 0.92 (m, 9); MS (ES+): 435. (M+1).

Example 1 Preparation of 4-Ethyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Ethyl pyridine-2-carboxylic acid HCl salt (Toronto) (117 mg, 0.64mmol) was suspended in dry acetonitrile (4 mL). Triethylamine (180 μL,1.28 mmol) was added and the reaction mixture was cooled to 0° C.Isobutyl chloroformate (129 μL, 0.62 mmol) was added and the reactionmixture was warmed to 4° C. After 1.5 h the activated ester solution wastransferred to a solution of 2b (R¹=Me, R²=Me), prepared as in Method C,in 1:1 acetone/water (2 mL) and warmed to 30° C. to dissolve.Triethylamine (80 μL, 0.057 mmol) was then added to the reactionmixture. The reaction mixture was stirred for 10 h at rt, thenevaporated to dryness and chromatographed on silica 94:5dichloromethane:0.25% ammonia in methanol to provide 11b (R¹=Me, R²=MeR³=H, R⁹=ethyl) (167 mg 69.7%).

MS (ESPOS): 385.2 [M+H].

A solution of pyridine 11b (m=2, R¹=Me, R²=Me R³=H, R⁹=ethyl) (167 mg,0.435 mmol) in 3:2 methanol/water (20 mL) was added to platinum(IV)oxide(339 mg, 0.521 mmol) in a Parr bottle. Concentrated HCl (52 μL, 0.52mmol) was then added. The bottle was purged, and charged with H₂ to 65psi and shaken for 24 h. The reaction mixture was filtered throughcelite and rinsed with methanol. The combined filtrate was evaporated todryness and chromatographed on silica 88:12 to 80:20 dichloromethane:0.25% ammonia in methanol to give 43 mg of a high R_(f) product and 49mg of a mixed fraction. Chromatography of the low R_(f) fraction onfluorosil 84:16 to 80:20 dichloromethane: 0.25% ammonia in methanolprovided the title compound (21.9 mg, 12.9%), which was taken up in 1:1acetonitrile:water (50 mL), 0.21μ millipore filtered, and cooled to 0°C. ¹N HCl (67 μL) in water (20 mL) was added and re-lyophilized toprovide the title compound HCl salt (24.0 mg) as a colorless powder.

¹H NMR (300 MHz, D₂O) δ 5.32 (d, J=5.8, 1), 4.14–4.06 (m, 1), 4.12 (s,2), 3.85 (d, J=3.30, 1), 3.60 (dd, J=3.3, 10.4, 1), 3.30 (dd, J=2.5,11.8, 1), 3.09 (m, 1), 2.56 (ddd, J=2.8, 12.9, 15.7, 1), 2.14 (s, 3),2.14–2.05 (m, 1), 1.96–1.90 (m, 1), 1.74–1.69 (m, 1), 1.45–1.35 (m, 1),1.33–1.23 (m, 2), 1.08–0.98 (m, 2), 0.86 (m, 9); MS(ESPOS): 391.4 [M+H],803.5.4 [2M+Na], (ESNEG): 389.5 [M−H].

Example 2 Preparation of 1-Methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-n-Propylhygric acid prepared by the method of Hoeksema, H. et. al.Journal of the American Chemical Society, 1967, 89 2448–2452 (157 mg,0.76 mmol) was suspended in dry acetonitrile (5 mL). Triethylamine (421μL, 3.02 mmol) was added and the reaction mixture was cooled to 0° C.Isobutyl chloroformate (98 μL, 0.76 mmol) was added and after 10 min thereaction was allowed to warm to 4° C. After 1.5 h a solution of 2b(R¹=Me, R²=Me), from Method C (190 mg, 0.76 mmol) in 1:1 acetone:water(5 mL) was added and the reaction mixture was stirred for 10 h at rt.The reaction mixture was evaporated to dryness and chromatographed onsilica 94:6 dichloromethane:0.25% ammonia in methanol. Fractions 14–18contained the product as a colorless oil (50.2 mg, 16.5%).

¹H NMR (300 MHz, D₂O) δ 5.33 (d, J=6.0, 1), 4.27–4.22 (m, 1), 4.18 (s,1), 4.09 (dd, J=5.8, 10.2, 1), 3.92–3.81 (m, 1), 3.92–3.81 (m, 1),3.64–3.59 (m, 1), 2.92 (s, 3), 2.92–2.85 (m, 1), 2.35–2.28 (m, 3), 2.13(s, 3), 1.46–1.41 (m, 2), 1.40–1.28 (m, 2), 0.89–0.84 (m, 9); MS(ESPOS):405.5 [M+H].

Example 3 Preparation of 1-methyl-4-propyl-pyrrolidine-2-carboxylic acid[3-cyano-2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Lincosamine 2b (R¹=Me, R²=CH₂CN) (54.2 mg, 0.20 mmol) prepared by MethodE was dissolved in DMF (0.7 mL). The reaction mixture was cooled to 0°C. and triethylamine (170 μL, 1.2 mmol) and BSTFA (96 μL, 0.36 mmol) wasadded. The reaction mixture was allowed to warm to rt, and stirred at rtfor 1 h. 4-n-Propylhygric acid prepared by the method of Hoeksema, etal., J. Am. Chem. Soc., 1967, 89 2448–2452 (66.4 mg, 0.32 mmol) and HATU(149 mg, 0.39 mmol) were added, and the mixture was stirred at rt for 3h. DMF was removed and the residue was dissolved in DCM (100 mL), washedwith saturated NaHCO₃ (30 mL) and brine (30 mL), and dried over sodiumsulfate. The residue obtained by removing the solvent was dissolved inmethanol (20 mL) and treated with Dowex™ resin H⁺ (300 mg) for 15 min.The crude product was eluted from the resin by washing with 5% TEA inMeOH (25 mL×15 min×2) and 5% TEA in MeCN (25 mL×15 min). The combinedeluent was evaporated to dryness and purified by silica gel columnchromatography using 7% 0.25M NH₃ in methanol in dichloromethane as theeluent to provide the title compound (24 mg, 28%).

¹H NMR (300 MHz, D₂O) δ 5.61 (d, J=5.8, 1), 4.59 (d, J=10.2, 1), 4.46(d, J=10.2, 1), 4.46 (dd, J=6.0, 10.4, 1), 4.05 (d, J=3.0, 1), 3.84 (dd,J=3.3, 10.4, 1), 3.48 (dd, J=5.8, 8.0, 1), 3.34 (dd, J=5.2, 10.2, 1)2.81–2.61 (m, 2), 2.65 (s, 3), 2.43 (s, 3), 2.31–2.10 (m, 2), 1.32 (d,J=6.0, 1), 1.18 (t, J=7.1, 3); MS(ESPOS): 430.5 [M+H] MS(ESPOS): 428.5[M−H].

Example 4 Preparation of 4-Ethyl-piperidine-2-carboxylic acid[2-hydroxy-2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Lincosamine 3b (R¹=Me, R²=Me) prepared by Method F (54.2 mg, 0.20 mmol)was dissolved in DMF (1.0 mL). The reaction mixture was cooled to 0° C.and triethylamine (178 μL, 1.3 mmol) and BSTFA (85 μL, 0.32 mmol) wereadded. The reaction mixture was allowed to warm to rt, and stirred atfor 1 h. 4-Ethyl pyridine-2-carboxylic acid HCl salt (Toronto) (55.3 mg,0.29 mmol) and HATU (122 mg, 0.32 mmol) were added, and the mixture wasstirred at rt for 3 h. DMF was removed and the residue was dissolved inTHF (10 mL), and treated with (600 mg) Amberlite A-26 F⁻ form resin andcatalytic TBAF for 5 h. The crude product was obtained by removal of theresin and evaporation of the solvent to dryness and purified by silicagel column chromatography using 10% 0.25M NH₃ in methanol indichloromethane as the eluent to provide the pyridine product 11b (m=2,R¹=Me, R²=Me R³=OH, R⁹=ethyl) (26 mg, 33%).

MS(ESNEG): 399.5 [M−H].

A solution of pyridine 11b (m=2, R¹=Me, R²=Me R³=OH, R⁹=ethyl) (26 mg,0.065 mmol) in 3:2 methanol:water (10 mL) was added to platinum(IV)oxide(51 mg) in a Parr bottle. Concentrated HCl (6.0 μL, 0.072 mmol) was thenadded. The bottle was purged and charged with H₂ to 65 psi and shakenfor 24 h. The reaction mixture was filtered through celite and rinsedwith methanol. The combined filtrate was evaporated to dryness andchromatographed on silica 80:20 dichloromethane: 0.25% ammonia inmethanol to give a high R_(f) product and the title compound (5.8 mg,21.8%).

¹H NMR (300 MHz, D₂O) δ 5.37 (d, J=6.0, 1), 4.41 (d, J=9.6, 1), 4.32 (d,J=9.3, 1), 4.08 (dd, J=6.6, 11.0, 1), 3.93–3.90 (m, 2), 3.59 (dd, J=3.0,10.7, 1), 3.93–3.90 (m, 2), 3.04 (apt dt, J=7.1, 14.6, 14.6, 1),2.24–2.18 (m, 1), 2.20 (s, 3), 1.70–1.60 (m, 1), 1.42–1.13 (m, 1), 0.88(dd, J=6.0, 7.4, 1); MS(ESPOS): 407.4 [M+H].

Example 5 Preparation of 1-Methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-hydroxyimino-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.041 mL, 0.28 mmol) and BSTFA (0.24 mL, 0.94 mmol) wereadded to the crude oxime 4b (R¹=Me, R⁷=H) prepared by Method G (50 mg,0.19 mmol) in DMF (3 mL) at 0° C. and the mixture was stirred at rtovernight. Next, 4-n-propylhygric acid (63 mg, 0.37 mmol) and HATU (142mg, 0.37 mmol) were added and the mixture was stirred at rt for 4 h. DMFwas removed and the residue was extracted with dichloromethane (100 mL)and washed with saturated bicarbonate (20 mL) and brine (20 mL). Theresidue obtained on removal of dichloromethane was then treated with 10%TFA in dichloroethane (10 mL) and dimethyl sulfide (0.5 mL) for 1 h. Thesolvent was then removed to obtain the crude product, which was purifiedby silica gel column chromatography using 20% methanol indichloromethane as the eluent to provide the title compound (20 mg,25%).

TLC: R_(f)=0.67 (20% methanol in dichloromethane); ¹H NMR (300 MHz,CD₃OD) δ 0.93 (t, J=6.8, 3), 1.31–1.44 (m, 4), 1.88 (s, 3), 1.99 (s, 3),2.09 (m, 2), 2.11 (m, 1), 2.62–2.98 (m, 3), 2.76 (s, 3), 3.60 (m, 2),4.10 (dd, J=5.7, 10.20, 1), 4.27 (d, J=9.6, 1), 5.23 (d, J=5.5, 1),MS(ESPOS): 420 (M+H).

Example 6 Preparation of 1-Methyl-4-propyl-pyrrolidine-2-carboxylic acid[2-methoxyimino-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

From crude oxime product 4b prepared by Method H (R¹=Me, R⁷=Me), thetitle compound was prepared as in Example 5 (10 mg, 47%).

TLC: R_(f)=0.55 (10% methanol in dichloromethane); ¹H NMR (300 MHz,CD₃OD) δ 0.91 (m, 3), 1.32 (m, 4), 1.88 (s, 3), 1.98 (s, 3), 1.78–2.04(m, 2), 2.34 (s, 3), 2.90 (dd, J=5.1, 6.30, 8.10 1), 3.21 (dd, J=6.3,10.2, 1), 3.57 (dd, J=3.3, 10.2, 1), 4.23 (dd, J=5.4, 10.2, 1), 5.25 (d,J=5.7, 1); MS(ESPOS): 434 (M+H).

Example 7 Preparation of 5-Butyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Lincosamine intermediate 2b (R¹=Me, R²=Me), prepared by Method C, wasdissolved in DMF (2 mL). Triethylamine (80 mg, 1 mmol) and BSTFA (307mg, 1.1 mmol) were added, and the mixture was stirred at rt for 1.5 h.Next, fusaric acid (143 mg, 0.7 mmol) and HATU (184 mg, 0.5 mmol) wereadded, and the mixture was stirred at rt for 3 h. DMF was removed andthe residue was dissolved in EtOAc (50 mL), washed with sodiumbicarbonate (10%, 30 mL) and brine (30 mL), and dried over sodiumsulfate. The residue obtained by removing the solvent was dissolved inmethanol and treated with Dowex™ resin H⁺ for 1 h. The crude productobtained by filtering the resin and removing the solvent was purified onsilica gel column chromatography using 10% methanol in dichloromethaneas the eluent to give the title compound (100 mg, 61%).

TLC R_(f)=0.6 (10% methanol in dichloromethane); ¹H NMR (300 MHz, CD₃OD)δ 8.47 (s, 1), 8.02 (d, J=8.1, 1), 7.80 (d, J=8.1, 1), 5.27 (d, J=5.4,1), 4.31 (m, 2), 4.12 (dd, J=5.7, 4.2, 1), 3.85 (d, J=3.0, 1), 3.56 (dd,J=3.3, 6.9, 1), 2.80 (m, 2), 2.24 (m, 1), 2.11 (s, 3), 1.67 (m, 2), 1.41(m, 2), 1.00 (m, 9); MS(ESPOS): 413 (M+H).

PtO₂ (50 mg, 0.22 mmol) was added to compound 11b (m=2, R¹=Me, R²=Me,R³=H, R⁹=butyl), (70 mg, 0.16 mmol) in methanol (2 mL), water (10 mL),and acetic acid (3 mL), and the mixture was hydrogenated at 50 psiovernight. The product obtained after filtering the catalyst andremoving the solvent was purified on silica gel column chromatographyusing 30% methanol in dichloromethane as the eluent (16 mg, 46%).

TLC R_(f)=0.7 (30% methanol in dichloromethane); ¹H NMR (300 MHz, CD₃OD)δ 5.24 (d, J=5.7, 1), 4.16 (m, 3), 3.82 (d, J=3.3, 1), 3.53 (m, 2), 2.93(m, 2), 2.09 (s, 3), 1.93 (m, 1), 1.76 (m, 2), 1.50 (m, 1), 1.30 (m, 7),0.92 (m, 9); MS(ESPOS): 419 (M+H).

Example 8 Preparation of 4-Pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.2 mL, 1.44 mmol, 3.6 equiv), followed by BSTFA (0.2 mL,0.76 mmol, 1.9 equiv), were added to a stirred suspension of 2b (R¹=Me,R²=Me) prepared by Method C (100.4 mg, 0.4 mmol, 1 equiv) in anhydrousDMF (2 mL) at 0° C. and under nitrogen. The resulting mixture wasstirred at 0° C. for 10 min, and then at rt for 50 min. The resultingsolution was cooled to 0° C. and a solution of 6c (R⁹=pentyl) (Scheme 6)prepared as described in Birkenmeyer, R. D; et al; Journal of MedicinalChemistry 1972, 15, 1255–1259. (144 mg, 0.51 mmol, 1.2 equiv) inanhydrous DMF (1.5 mL) was added, followed by solid HATU. The reactionmixture was allowed to warm to rt, and after 2 h, the reaction solutionwas evaporated to dryness under vacuum. The residual oil obtained wasdiluted with EtOAc (150 mL), washed sequentially with 10% citric acid(2×30 mL), 1:1 saturated aqueous NaHCO₃, water (2×30 mL), and brine (30mL), dried over Na₂SO₄, and evaporated to dryness.

1,2-Dichloroethane (8 mL), followed by dimethyl sulfide (180.3 μl), TFA(2.7 mL), and water (180.3 μl) were added to the crude product (267.5mg) obtained above. The resulting mixture was stirred at rt for 1 h andevaporated to a minimal volume, diluted with DCE (3×30 mL), andevaporated to dryness. The residue obtained was purified bychromatography over silica gel, with a gradient eluent of 8–10% methanolammonia in dichloromethane. The desired fractions were pooled together,evaporated to dryness, and lyophilized to furnish the title compound asa white fluffy powder (35.6 mg, 21.2%).

TLC, R_(f)=0.15 (16% 0.25M methanolic ammonia in dichloromethane). ¹HNMR (300 MHz, D₂O) δ 5.4 (d, J=5.8, 1), 3.91(s, 1), 3.69–3.66 (m, 3),2.1 (s, 3), 1.32–1.15 (m, 3.37), 0.93–0.87 (m, 9.8); MS(ESPOS): 419.5[M+H], (ESNEG): 417.45 [M−H].

Example 9 Preparation of 4-(3-Methyl-butyl)-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.13 mL, 0.96 mmol, 3.2 equiv), followed by BSTFA (0.12mL, 0.45 mmol, 1.5 equiv), were added to a solution of 2b (R¹=Me, R²=Me)prepared by Method C (75 mg, 0.30 mmol, 1 equiv) in dry DMF (0.8 mL) at0° C. The reaction mixture was stirred at 0° C. for 10 min, and then atrt for 50 min. To the reaction mixture was added acid 7d(R⁹=2-methylbutyl), prepared by method J (160 mg, 0.56 mmol, 1.9 equiv),in a 25 mL round-bottom flask. Then HATU was added (227 mg, 0.60 mmol, 2equiv). The reaction mixture was stirred at rt for 3 h. The reactionmixture was evaporated to dryness, taken up in ethyl acetate (100 mL),washed with 10% citric acid (2×60 mL), water (60 mL), half sat. NaHCO₃(60 mL), and brine. The organic layer was dried over Na₂SO₄ andevaporated to give a yellow syrup.

Trifluoroacetic acid (5 mL) and water (0.33 mL) were added to a solutionof the above syrup in dichloromethane (15 mL) with methyl sulfide (0.33mL). The reaction mixture was stirred at rt for 1 h. The solvent wasremoved under vacuum and co-evaporated with toluene twice. The residuewas purified by chromatography to provide the title compound (75 mg,60%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.4, 1), 4.18–3.99 (m, 4), 3.75 (d,J=2.4, 1), 3.51 (dd, J=3.3, 10.5, 1), 3.38–3.31 (m, 1), 2.68 (dd, J=8.2,10.6, 1), 2.23–2.05 (m, 3), 2.10 (s, 3), 1.97–1.87 (m, 1), 1.59–1.47 (m,1), 1.46–1.34 (m, 2), 1.25–1.16 (m, 2), 0.92–0.88 (m, 12). MS(ESPOS):419.5 [M+H]⁺, MS(ESNEG): 417.5 [M−H]⁻.

Example 10 Preparation of 4-Pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.6 mL, 4.33 mmol, 3.6 equiv), followed by BSTFA (0.6 mL,2.27 mmol, 1.9 equiv), were added to a stirred suspension of 2b (R¹=Me,R²=Me) prepared by Method C (298.8 mg, 1.19 mmol, 1 equiv) in anhydrousDMF (5 mL) at 0° C. and under nitrogen. The resulting mixture wasstirred at 0° C. for 10 min, and then at rt for 50 min. The resultingsolution was cooled to 0° C. and a solution of 7d (R⁹=n-pentyl) wasprepared by Method K (400.1 mg, 1.40 mmol, 1.2 equiv) in anhydrous DMF(5 mL) was added, followed by solid HATU (678.7 mg, 1.79 mmol, 1.5equiv). The reaction mixture was allowed to warm to rt and after 2 h thereaction solution was evaporated to dryness under vacuum. The residualoil obtained was diluted with EtOAc (400 mL), washed sequentially with10% citric acid (2×100 mL), 1:1 saturated aqueous NaHCO₃, water (2×100mL), and brine (100 mL), dried over Na₂SO₄, and evaporated to dryness.

1,2-Dichloroethane (35 mL), followed by dimethylsulfide (768 μL), TFA(11.5 mL), and water (768 μL) were added to the crude product (1.14 g)obtained above. The resulting mixture was stirred at rt for 1 h,evaporated to a minimal volume, diluted with DCE (3×90 mL), andevaporated to dryness. One-third of the residue obtained was purified bychromatography over silica gel, with a gradient eluent of 8–12% methanolammonia in dichloromethane. The desired fractions were pooled together,evaporated to dryness, treated with deuterium oxide/anhydrousacetonitrile, and lyophilized to furnish a white fluffy powder (68.2 mg,41.1%); TLC, R_(f)=0.15 {16% 0.25M methanolic ammonia indichloromethane}. ¹H NMR (300 MHz, D₂O) δ 5.41 (d, J=5.8, 1H), 4.55 (m,1), 4.24 (s, 2), 4.14 (m, 1), 3.91 (d, J=3.3, 1), 3.70–3.66 (m, 2), 3.15(m, 1), 2.36–2.27 (m, 2), 2.19 (s, 5), 1.59–1.13 (m, 9H), 0.93–0.88 (m,9); ¹³C NMR (D₂O,): δ 170.4, 119.4, 88.4, 70.9, 69.3, 68.8, 68.2, 60.0,53.4, 51.4, 37.3, 36.7, 31.3, 27.9, 27.2, 22.3, 20.1, 14.8, 13.7, 13.3;MS(ESPOS):419.6[M+H];(ESNEG):417.5[M−H].

Example 11 Preparation of 1-Methyl-4-propyl-pyrrolidine-2-carboxylicacid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

30% Trifluoroacetic acid in dichloroethane (10 mL) and dimethylsulfide(0.5 mL) were added to lincosamine intermediate 5b (R¹=Me), prepared byMethod H (100 mg, 0.20 mmol). The mixture was stirred at rt for 1 h. Thesolvent was removed and the residue was kept under high vacuum for 1 h.N-Methyl-4-trans-propylproline (53 mg, 0.4 mmol) and HATU (114 mg, 0.30mmol) were added to the dried compound in DMF (3 mL), and the mixturewas stirred at rt overnight. DMF was removed and the residue obtainedwas then extracted with ethyl acetate (100 mL) and washed with saturatedbicarbonate (50 mL). The organic portion was then dried using magnesiumsulfate and the solvent was removed to obtain the crude product. Thecrude product was purified on silica gel column using ethyl acetate asthe eluent (50 mg, 46%). The product (50 mg, 0.09 mmol) was then takenin methanol (2 mL) and water (1 mL), to which solid potassium carbonate(124 mg, 0.90 mmol) was added and the mixture was stirred at rt for 24h. Solvents were then removed and the crude product was purified onsilica gel column using 20% methanol in dichloromethane as the eluent(20 mg, 52%).

TLC: R_(f)=0.57 (20% methanol in dichloromethane); MS(ESPOS): 427 (M+H).¹H NMR (300 MHz, CD₃OD) δ 0.91 (m, 3), 1.34 (m, 4), 1.69 (t, J=19.8, 3),1.98 (s, 3), 2.20 (m, 2), 2.46 (s, 3), 3.18 (dd, J=5.1, 10.20, 1), 3.93(d, J=3.0, 1), 4.08 (dd, J=3.3, 10.20, 1), 4.40–4.70 (m, 2), 5.28 (d,J=5.4, 1).

Example 12 Preparation of 4-Pentyl-pyrrolidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Boc 4-trans-Pentylproline 7d (R⁹=n-pentyl) (179 mg, 0.631 mmol), HATU(299 mg, 0.789 mmol), and diethylisopropylamine (182 mg, 1.2 mmol) wereadded to lincosamine intermediate 5b (R¹=Me) prepared by method I (210mg, 0.526 mmol) in DMF (3 mL) at 0° C. The mixture was stirred at rtovernight. After removing DMF, the residue was taken in ethyl acetateand washed with saturated bicarbonate (30 mL). The organic portion wasthen dried over sodium sulfate and the solvent was removed to obtain thecrude product. The crude product was purified by column chromatographyusing 30% ethyl acetate in hexanes as the eluent (200 mg, 57%).Potassium carbonate (450 mg, 3.0 mmol) was added to the product (200 mg,0.30 mmol) of the above reaction in methanol (3 mL) and water (1 mL),and the mixture was stirred at rt for 2 h. The solvent was removed andthe residue obtained was taken in 30% trifluoroacetic acid indichloroethane (10 mL) and dimethyl sulfide (0.5 mL) and stirred for 1h. After removing the solvent, the crude product obtained was purifiedby column using 10% methanol in dichloromethane as the eluent (10 mg,90%).

TLC: R_(f)=0.56 (20% methanol in dichloromethane); ¹H NMR (300 MHz,CD₃OD) δ 0.90 (m, 3), 1.31 (m, 7), 1.44 (m, 20), 1.73 (t, J=19.5,3),2.02 (m, 1), 2.08 (s, 3), 2.24 (m, 2), 2.89 (t, J=9.9, 1), 3.56 (m, 2),3.86 (s, 1), 4.07 (dd, J=6.0, 9.6, 1), 4.37 (m, 2), 4.63 (m, 1), 5.28(d, J=5.4, 1); MS(ESPOS): 441 (M+H).

Example 13 Preparation of4-[3-(4-Fluoro-phenyl)-propyl]-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.13 mL, 0.96 mmol, 3.2 equiv), followed by BSTFA (0.12mL, 0.45 mmol, 1.5 equiv), were added to a solution of 2b (R¹=Me, R²=Me)prepared by Method C (75 mg, 0.30 mmol, 1 equiv) in dry DMF (0.8 mL) at0° C. The reaction mixture was stirred at 0° C. for 10 min, and then atrt for 50 min. The reaction mixture was added to acid 8c(R⁹=3-(4-fluorophenyl)propyl) prepared by Method L (120 mg, 0.34 mmol,1.1 equiv) in a 25 mL round bottom flask, followed by HATU (160 mg, 0.42mmol, 1.4 equiv). The reaction mixture was stirred at rt for 3 h. Thereaction mixture was evaporated to dryness, taken up in ethyl acetate(100 mL), and washed with 10% citric acid (2×60 mL), water (60 mL), halfsat. NaHCO₃ (60 mL), and brine. The organic layer was dried over Na₂SO₄and evaporated to give a yellow syrup.

Trifluoroacetic acid (5 mL) and water (0.33 mL) were added to a solutionof the above syrup in dichloromethane (15 mL) with methyl sulfide (0.33mL). The reaction mixture was stirred at rt for 1 h. The solvent wasremoved under vacuum and co-evaporated with toluene twice. The residuewas purified by chromatography to provide the title compound (90 mg,62%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 7.94 (brs, 1), 7.11–7.06 (m, 2), 6.97–6.90 (m,2), 5.31 (d, J=5.4, 1), 4.10 (dd, J=5.7, 9.9, 1), 3.96–3.82 (m, 3),3.68–3.52 (m, 2), 3.10–3.20 (m, 1), 2.70–2.60 (m, 1), 2.56 (dd, J=7.4,7.4, 2), 2.36–2.24 (m, 1), 2.13 (s, 3), 2.10–1.93 (m, 2), 1.85–1.73 (m,1), 1.64–1.50 (m, 2), 1.40–1.30 (m, 2), 0.92–0.85 (m, 6). MS(ESPOS):485.5 [M+H]³⁰ . MS(ESNEG): 483.5 [M−H]⁻.

Example 14 Preparation of4-(3,3-Difluoro-propyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.13 mL, 0.96 mmol, 3.2 equiv), followed by BSTFA (0.12mL, 0.45 mmol, 1.5 equiv), were added to a solution of 2b (R¹=Me, R²=Me)prepared by Method C (75 mg, 0.30 mmol, 1 equiv) in dry DMF (0.8 mL) at0° C. The reaction mixture was stirred at 0° C. for 10 min, and then atrt for 50 min. The reaction mixture was added to acid 8c(R⁹=3,3-difluoropropyl) prepared by-Method N (97 mg, 0.33 mmol, 1.1equiv) in a 25 mL round bottom flask, followed by HATU (170 mg, 0.45mmol, 1.5 equiv). The reaction mixture was stirred at rt for 3 h. Thereaction mixture was evaporated to dryness, taken up in ethyl acetate(100 mL), and washed with 10% citric acid (2×60 mL), water (60 mL), halfsat. NaHCO₃ (60 mL), and brine. The organic layer was dried over Na₂SO₄and evaporated to give a yellow syrup.

Trifluoroacetic acid (5 mL) and water (0.33 mL) were added to a solutionof the above syrup in dichloromethane (15 mL) with methyl sulfide (0.33mL). The reaction mixture was stirred at rt for 1 h. The solvent wasremoved under vacuum and co-evaporated with toluene twice. The residuewas purified by chromatography to provide the title compound (81 mg,64%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 7.92 (d, J=8.1, 1), 5.80 (dddd, J=4.2, 4.2,57, 57, 1), 5.31 (d, J=5.7, 1), 4.11 (dd, J=5.4, 9.9, 1), 3.96–3.82 (m,3), 3.64–3.52 (m, 2), 3.23–3.10 (m, 1), 2.73–2.60 (m, 1), 2.36–2.23 (m,1), 2.13 (s, 3), 2.18–1.95 (m, 2), 1.90–1.73 (m, 3). 1.56–1.43 (m, 2),0.93–0.85 (m, 6). MS(ESPOS): 427.5 [M+H]⁺, MS(ESNEG): 425.5 [M−H]⁻.

Example 15 Preparation of4-[3-(4-Chloro-phenyl)-propyl]-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (88.3 μL, 0.64 mmol, 3.2 equiv), followed by BSTFA (79.2mL, 0.30 mmol, 1.5 equiv), were added to a solution of 2b (R¹=Me, R²=Me)prepared by Method C (50 mg, 0.20 mmol, 1 equiv) in dry DMF (0.5 mL) at0° C. The reaction mixture was stirred at 0° C. for 10 min, and then atrt for 50 min. The reaction mixture was added to 8c(R⁹=3-(4-chlorophenyl)propyl) prepared by Method M (97.3 mg, 0.26 mmol,1.3 equiv) in a 25 mL round bottom flask, followed by HATU (123 mg, 0.32mmol, 1.6 equiv). The reaction mixture was stirred at rt for 3 h. Thereaction mixture was evaporated to dryness, taken up in ethyl acetate(60 mL), and washed with 10% citric acid (2×40 mL), water (40 mL), halfsat. NaHCO₃ (40 mL), and brine. The organic layer was dried over Na₂SO₄and evaporated to give a yellow syrup.

Trifluoroacetic acid (3 mL) and water (0.2 mL) were added to a solutionof the above syrup in dichloromethane (9 mL) with methyl sulfide (0.2mL). The reaction mixture was stirred at rt for 1 h. The solvent wasremoved under vacuum and co-evaporated with toluene twice. The residuewas purified by chromatography to provide the title compound (41.6 mg,42%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 7.26–7.21 (m, 2), 7.17–7.12 (m, 2), 5.23 (d,J=5.7, 1), 4.10–4.00 (m, 3), 3.83–3.75 (m, 1), 3.74–3.70 (m, 1),3.54–3.48 (m, 1), 3.25–3.18 (m, 1), 2.63–2.50 (m, 3), 2.20–2.00 (m, 3),2.09 (s, 3), 1.85–1.74 (m, 1), 1.68–1.55 (m, 2), 1.42–1.33 (m, 2),0.95–0.85 (m, 6). MS(ESPOS): 501.5 [M+H]⁺ MS(ESNEG): 499.4 [M−H]⁻.

Example 16 Preparation of4-(2,2-Difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (88.3 mL, 0.64 mmol, 3.2 equiv), followed by BSTFA (79.2mL, 0.30 mmol, 1.5 equiv), were added to a solution of 2b (R¹=Me, R²=Me)prepared by Method C (50 mg, 0.20 mmol, 1 equiv) in dry DMF (0.5 mL) at0° C. The reaction mixture was stirred at 0° C. for 10 min, and then atrt for 50 min. The reaction mixture was added to the acid 9d(R⁹=2,2-difluoropentyl) prepared by Method O (67.7 mg, 0.21 mmol, 1.1equiv) in a 25 mL round bottom flask, followed by HATU (101 mg, 0.27mmol, 1.3 equiv). The reaction mixture was stirred at rt for 3 h. Thereaction mixture was evaporated to dryness, taken up in ethyl acetate(60 mL), and washed with 10% citric acid (2×40 mL), water (40 mL), halfsat. NaHCO₃ (40 mL), and brine. The organic layer was dried over Na₂SO₄and evaporated to give a yellow syrup.

Trifluoroacetic acid (3 mL) and water (0.20 mL) were added to a solutionof the above syrup in dichloromethane (9 mL) with methyl sulfide (0.20mL). The reaction mixture was stirred at rt for 1 h. The solvent wasremoved under vacuum and co-evaporated with toluene twice. The residuewas purified by chromatography to provide the title compound (56 mg,62%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.17–4.04 (m, 3), 3.98(dd, J=3.3,9.3, 1), 3.77 (d, J=3, 1), 3.51 (dd, J=3.4, 10.3, 1), 3.40(dd, J=6.9, 10.5, 1), 2.71 (dd, J=10.2, 10.2, 1), 2.42–2.33 (m, 1),2.23–2.11 (m, 2), 2.10 (s, 3), 2.08–1.73 (m, 5), 1.56–1.42 (m, 2),0.99–0.89 (m, 9). MS(ESPOS): 455.5 [M+H]⁺; MS(ESNEG): 453.5 [M−H]⁻.

Example 17 Preparation of 4-Propyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.18 mL, 1.26, mmol) and BSTFA (0.549 mL, 2.1 mmol) wereadded to the lincosamine intermediate 2b (R¹=Me, R²=Me) prepared byMethod D (102 mg, 0.42 mmol) in DMF (5 mL) at 0° C., and the mixture wasstirred at rt for 3. h. Acid 10b (R⁹=propyl) prepared by Method P (200mg, 0.84 mmol) and HATU (319 mg, 0.84 mmol) were added and the mixturewas stirred for 4 h at rt. DMF was removed and the residue was extractedwith ethyl acetate (100 mL) and washed with saturated bicarbonate (40mL). The product obtained by removal of solvent was taken up in methanoland treated with Dowex™ H⁺ resin for 1 h. After filtering the resin,methanol was removed to obtain the crude product. The crude product wasthen purified on silica gel column using 10% methanol in dichloromethaneas the eluent to provide pyridine 11b (R¹=Me, R²=Me, R³=H, R⁹=propyl)(117 mg, 58%).

TLC: R_(f)=0.81 (10% methanol in dichloromethane); ¹H NMR (300 MHz,CDCl₃) δ 1.20 (t, J=6.3, 6H), 2.19 (m, 2), 2.32 (s, 3), 2.43 (m, 1),2.84–2.97 (m, 4), 3.74 (m, 1), 4.06 (m, 1), 4.31 (m, 1), 4.52 (m, 2),5.42 (d, J=5.7, 1), 7.33–7.61 (m, 5), 7.80 (m, 1), 8.15 (s, 1), 8.69 (d,J=4.8, 1); MS (ESPOS): 475 (M+H).

PtO₂ (100 mg, 0.44 mmol) was added to pyridine 11b (R¹=Me, R²=Me, R³=H,R⁹=propyl), (150 mg, 0.37 mmol) in methanol (2 mL), water (10 mL), andacetic acid (3 mL), and the mixture was hydrogenated at 50 psiovernight. The product obtained after filtering the catalyst andremoving the solvent was purified by silica gel column chromatographyusing 30% methanol in dichloromethane as the eluent to provide the titlecompound (20 mg, 14%).

TLC: R_(f)=0.7 (50% methanol in dichloromethane); ¹H NMR (300 MHz,CD₃OD) δ 5.24 (d, J=6.9, 1), 4.86 (m, 1), 4.13 (m, 2), 3.79 (d, J=3.3,1), 3.52 (dd, J=3.3, 9.9, 1), 3.32 (m, 1), 3.17 (m, 1), 2.67 (m, 1),2.17 (m, 1), 2.10 (s, 3), 1.97 (m, 1), 1.74 (m, 1), 1.54 (m, 1), 1.38(m, 2), 1.31 (m, 2), 1.14 (m, 2), 1.02 (m, 9) MS(ESPOS): 405 (M+H).

Example 18 Preparation of1-(2-Hydroxy-ethyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

DIEA (0.1 mL, 0.57 mmol) and liquid ethylene oxide (3 mL) were added toa stirred solution of crude 4-pentyl-pyrrolidin-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-(methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]amide,prepared as in Example 10 (237.4 mg), in anhydrous methanol (10 mL), at0° C. and under nitrogen. The resulting solution was stirred at −4° C.for 18 h and evaporated to dryness. The residue obtained was purified bychromatography over silica gel with an eluent of 5% methanolic ammoniain dichloromethane. The desired fractions were evaporated and the reiduelyophilized (deuterium oxide/anhydrous acetonitrile, 1:1, v/v, 10 mL) tofurnish the title compound as a fluffy white powder (50.1 mg, 30.2%);TLC, R_(f)=0.68 (14% methanolic ammonia in dichloromethane); ¹H NMR (300MHz) δ 5.40 (d, J=5.8, 1), 4.55 (m, 1), 4.24 (s, 1), 4.17–4.11 (m, 1)3.99–3.89 (m, 4), 3.69–3.65 (m, 1), 3.47 (d, J=4.4, 2), 3.01 (m, 1),2.33 (br s, 4), 2.18 (s, 4), 1.57–1.32 (m, 9), 0.94–0.87 (m, 9).

MS(ESPOS):464[M+H]; (ESNEG):497.5[M−H+HCl].

Example 19 Preparation of1-(2-Hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

DIEA (0.1 mL, 0.58 mmol, 1 equiv) and R(+)-propylene oxide (3 mL) wereadded to a stirred cool solution of crude4-pentyl-pyrrolidin-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-(methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]amide (307.6 mg, 0.58 mmol, 1 equiv), prepared as in Example 10, inanhydrous methanol (10 mL), at 0° C. and under nitrogen. The resultingsolution was stirred at 4° C. for 18 h and evaporated to dryness. Theresidue obtained was purified by chromatography over silica gel, with aneluent of 6% methanolic ammonia in dichloromethane. The desiredfractions were evaporated, and lyophilized (deuterium oxide/anhydrousacetonitrile, 1:1, v/v, 20 mL) to furnish the title compound as a fluffywhite powder (91 mg, 48%).

TLC, R_(f)=0.7. (14% methanolic ammonia in dichloromethane); ¹H NMR(300MHz, CD₃OD) δ 5.44 (d, J=5.5, 1), 4.31 (s, 2), 4.26–4.11 (m, 1), 3.97(d, J=3.3,1.1,1), 3.75 (dd, J=3.3,3.3,1), 3.39 (dd, J=3.8,3.8,1), 2.31(s, 3), 1.5–0.95 (m, 12), 1.34 (d, J=6.0, 4), 1.17–1.10 (m, 13);MS(ESPOS): 477.6 [M+H], (ESNEG): 475.6 [M−H].

Example 20 Preparation of1-(2-Hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Dimethyl sulfide (62 μL), TFA (1 mL), and water (62 μL) were added to astirred solution of the Boc-protected 4-pentyl-pyrrolidin-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-(methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide(92mg, 0.18 mmol), prepared as in Example 10, in anhydrousdichloroethane (3 mL). The resulting solution was stirred at rt for 1 hand evaporated to dryness. Anhydrous methanol (8 mL) and DIEA (31 μL,0.18 mmol) were added to the residue obtained. The mixture was cooled to−4° C. and S-(−)-propylene oxide (2 mL) was added. The resultingsolution was stirred at −4° C. for 18 h, evaporated to dryness, andpurified by chromatography over silica gel, with an eluent of 6%methanolic ammonia in dichloromethane. The desired fractions wereevaporated and lyophilized (deuterium/anhydrous acetonitrile, 1:1, v/v,8 mL) to furnish the title compound as a fluffy white powder (29.8 mg,31.2%).

TLC, R_(f)=0.7 (12% methanolic ammonia in dichloromethane); 1H NMR (300MHz, CD₃OD) δ 5.44 (d, J=5.5,1), 4.35–4.19 (m, 4), 4.02 (d, J=3.3, 2),2.75 (d, J=6.3,2,2,3), 2.3 (s, 3), 1.50 (m, 11), 1.4 (d, J=6.0, 3.5, 3),1.16–1.10 (m, 12). MS(ESPOS): 477.6 [M+H]; (ESNEG) 475.4 [M−H].

Example 21 Preparation of1-(3-Hydroxy-propyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Triethylamine (0.2 mL, 1.38 mmol, 3 equiv), followed by3-bromo-1-propanol (60 μL, 0.69 mmol, 1.5 equiv), were added to astirred solution of crude 4-pentyl-pyrrolidin-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-(methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide(192.5 mg, 0.46 mmol, 1 equiv), prepared as in Example 10, in anhydrousacetonitrile (2 mL), under nitrogen. The resulting mixture was stirredat rt for 18 h and evaporated to dryness. The residue obtained waspurified by chromatography over silica gel with an eluent of 5%methanolic ammonia in dichloromethane. The desired fractions were pooledtogether, evaporated to dryness, and lyophilized to furnish the titlecompound as a white fluffy powder (13.5 mg, 6%).

TLC, R_(f)=0.75 (14% methanolic ammonia in dichloromethane); ¹H NMR (300MHz, CD₃OD) δ 5.44 (d, J=5.8, 1), 4.33–4.26 (m, 4), 4.01 (d, J=2.7, 1),3.85–3.74 (m, 6), 2.29 (s, 3), 2.1 (m, 4), 1.54 (m, 8), 1.16–1.08 (m,12); MS (ESPOS): 477.6 [M+H].

Example 22 Preparation of1-(2-Hydroxy-ethyl)-4-(3-methyl-butyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Ethylene oxide (0.6 mL) was added to a solution of4-(3-methyl-butyl)-pyrrolidin-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-(methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide(35.1 mg, 0.084 mmol), prepared as in Example 9, in methanol (3 mL), at0° C. The reaction mixture was stirred at 4° C. overnight. Additionalethylene oxide (0.6 mL) was added and stirred at 4° C. overnight. Thereaction mixture was concentrated and purified by chromatography to givethe title compound as a white solid (24.1 mg, 62%).

¹H NMR (300 MHz, CDCl₃) δ 7.68 (d, J=9.0, 1), 5.32 (d, J=5.4, 1), 5.24(d, J=3.0, 1), 4.13–4.07 (m, 1), 4.01 (ddd, J=2.8, 9.9, 9.9, 1), 3.86(d, J=10.8, 1), 3.78–3.68 (m, 2), 3.61–3.57 (m, 1), 3.56–3.48 (m, 1),3.36–3.32 (m, 1), 3.27–3.21 (m, 1), 2.94–2.85 (m, 1), 2.76–2.70 (m, 1),2.55 (ddd, J=3.6, 3.6, 12.6, 1), 2.41–2.37 (m, 1), 2.36–2.27 (m, 1),2.15 (s, 3), 2.03–1.95 (m, 2), 1.93–1.81 (m, 1), 1.54–1.42 (m, 1),1.39–1.26 (m, 2), 1.22–1.10 (m, 2), 0.99–0.92 (m, 6), 0.90–0.84 (m, 6).MS(ESPOS): 463.5 [M+H]⁺ MS(ESNEG): 461.5 [M−H]⁻.

Example 23 Preparation of4-(3,3-Difluoro-propyl)-1-(2-hydroxy-ethyl)-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Ethylene oxide (0.4 mL) was added to a solution of1-[2-(S)-4-(R)-(3,3-difluoroprop-1-yl)pyrrolidin-2-yl]-N-{1-(R)-[2-(S)-3-(S),4-(S),5-(R)-trihydroxy-6-(R)-(methylthio)tetrahydropyran-2-yl]-2-methylprop-1-yl}acetamide,prepared as in Example 14 (29.7 mg, 0.07 mmol), in methanol (2 mL), at0° C. The reaction mixture was stirred at 4° C. overnight. Additionalethylene oxide (0.4 mL) was added and stirred at 4° C. overnight. Thereaction mixture was concentrated and purified by chromatography to givea white solid,1-[2-(S)-4-(R)-(3-methylbut-1-yl)-N-(2-hydroxyeth-1-yl)pyrrolidin-2-yl]-N-{1-(R)-[2-(S)-3-(S),4-(S),5-(R)-trihydroxy-6-(R)-(methylthio)tetrahydropyran-2-yl]-2-methylprop-1-yl}acetamide(19.3 mg, 59%).

¹H NMR (300 MHz, CDCl₃) δ 5.86 (dddd, J=4.3,4.3, 57, 57, 1), 5.23 (d,J=5.7, 1), 4.13–4.04 (m, 3), 3.75 (d, J=3.3, 1), 3.73–3.57 (m, 2), 3.53(dd, J=3.3, 10.2, 1), 3.42–3.36 (m, 1), 3.26–3.18 (m, 1), 2.88–2.78 (m,1), 2.62–2.55 (m, 1), 2.17–2.00 (m, 4), 2.10 (s, 3), 1.94–1.73 (m, 3),1.55–1.45 (m, 2), 0.98–0.91 (m, 6).

MS (ESPOS): 471.5 [M+H]⁺, MS (ESNEG): 469.4 [M−H]⁻.

Example 24 Preparation of1-(2-Hydroxy-ethyl)-4-pentyl-pyrrolidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

Ethylene oxide (1 mL, excess) was added to the title compound fromExample 12 (60 mg, 0.136 mmol) in methanol (5 mL) at 0° C., and themixture was stirred at 4° C. overnight. The solvent was removed and thecrude product was purified by silica gel column chromatography using 10%methanol in dichloromethane as the eluent (25 mg, 38%).

TLC: R_(f)=0.76 (5% methanol in dichloromethane); ¹H NMR (300 MHz,CD₃OD) δ 0.89 (m, 3), 1.30 (m, 8), 1.68 (t, J=19.4, 3), 1.78 (m, 1),1.99 (m, 2), 2.07 (s, 3), 2.63 (m, 1), 2.73 (m, 1), 3.19 (m, 1), 3.58(m, 3), 3.95 (m, 1), 4.08 (dd, J=6.0, 9.90, 1), 4.44–4.60 (m, 2), 5.26(d, J=5.4, 1); MS (ESPOS): 485 (M+H).

Example 25 Preparation of 4-Pentyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Pentylpyridine-2-carboxylic acid (10b) (R⁹=pentyl) was made byemploying general Method P. To 4-pentylpyridine (3 g, 20 mmol) in aceticacid (30 mL), hydrogen peroxide (0.7 g, 30%, 20 mmol) was added andrefluxed overnight. Removal of solvent resulted in residue which wasdissolved in DCM (100 mL) dried over MgSO₄ and filtered. Removal of DCMresulted in a brown liquid, 4-pentylpyridine-N-oxide, (3.3 g, 100%). Totrimethylsilyl cyanide (2.37 g, 24 mmol), 4-pentylpyridine N-oxide (3.3g, 20 mmol) in DCM (10 mL) was added followed by dropwise addition ofdimethylcarbamoyl chloride (2.56 g, 24 mmol) in DCM (10 mL). Afterstirring at room temperature overnight, sodium bicarbonate (100 mL, 10%)was added and the organic layer was separated. The aqueous layer wasextracted twice with DCM (50 mL) and the combined organic layer wasdried over magnesium sulfate. Removal of solvent resulted in compound10a (R⁹=pentyl) (4.1 g, 100%).

¹H NMR (300 MHz, CD₃OD) δ 8.52 (m, 1), 7.46 (s, 1), 7.27 (m, 1), 3.00(m, 2), 2.60 (m, 2), 1.60 (m, 2), 1.27 (m, 2), 0.86 (m, 3). MS (ES+):175 (M+1).

The 4-Pentyl-2-cyanopyridine (10a) (R⁹=pentyl) (3.4 g, 19.5 mmol) fromthe previous step was dissolved in HCl (6 N, 100 mL) and refluxedovernight. The residue obtained on removal of HCl was purified by columnchromatography using 20% MeOH in DCM (3.7 g, 100%) to give productcompound 10(b). ¹H NMR (300 MHz, CD₃OD) δ 8.74 (d, J=6.3, 1), 8.39 (s,1), 8.06 (d, J=6.3, 1), 2.98 (t, J=7.2, 2), 1.77(m, 2), 1.39 (m, 4),0.95 (t, J=7.2, 3H). MS (ES−): 192 (M−1).

Then to 7-methyl α-thiolincosaminide 2b (R¹=Me, R²=Me) (90 mg, 0.35mmol) in DMF (2 ml), TEA (72 mg, 0.7 mmol), BSTFA (276 mg, 1.05 mmol)were added at 0° C. and stirred at room temperature for 1.5 hr. Then theacid (10b) (R⁹=pentyl) (138 mg, 0.7 mmol) and HATU (165 mg, 0.53 mmol)was added to the reaction mixture, and stirred at room temperatureovernight. DMF was completely removed, the residue was taken up in EtOAc(50 mL), washed with sodium bicarbonate (10%, 50 mL), brine (50 mL). Theproduct obtained after drying over magnesium sulfate and concentrationwas taken up in methanol (10 mL) and treated with resin (150 mg) for 3hr. The resin was filtered and the solvent was removed. Purification ofthe crude product was carried out silica gel column chromatography using3% MeOH in DCM as eluent to obtain compound 11b (R¹=Me, R²=Me R³=H,R⁹=pentyl) (90 mg, 59%):

¹H NMR (300 MHz, CD₃OD) δ 8.50 (d, J=5.1, 1), 7.95 (s, 1), 7.11 (m, 1),5.25 (d, J=5.7, 1), 4.20–4.87 (m, 3), 3.85 (d, J=3.3, 1), 3.55 (dd,J=3.3, 7.2, 1), 2.72 (m, 2), 2.16 (m, 4), 1.67 (m, 2), 1.35 (m, 4), 0.96(m, 9). MS (ES+): 427 (M+1).

To pyridine 11b (R¹=Me, R²=Me R³=H, R⁹=pentyl) (90 mg, 0.7 mmol) inwater (10 mL), AcOH (3 mL) and MeOH (2 mL), PtO₂ (100 mg) was added,hydrogenated at 55 psi overnight. The solvent was removed to obtain thecrude product. Purification of the crude product was carried out bysilica gel column chromatography using 20% MeOH in DCM to obtain thetitle compound (35 mg, 38%).

¹H NMR (300 MHz, CD₃OD) δ 5.23 (d, J=5.1, 1), 4.17 (m, 3), 3.79 (d,J=3.3, 1), 3.52 (m, 1), 3.38 (m, 1), 3.07 (m, 1), 2.68 (m, 1), 2.14 (m,4), 1.88 (m, 1), 1.71 (m, 1), 1.52 (m, 1), 1.30 (m, 8), 1.07 (m, 3),0.90 (m, 9); MS (ES+): 433 (M+1).

Example 26 Preparation of 4-Methoxy-pipreridine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Methoxypyridine-2-carboxylic acid, compound 10(b) (R⁹=methoxy), wasmade employing general Method P. To trimethylsilyl cyanide (0.95 g, 9.6mmol), 4-methoxypyridine N-oxide (1 g, 8 mmol) in DCM (10 mL) was added,followed by dimethylcarbamoyl chloride (1.03 g, 9.6 mmol) in DCM (10mL), dropwise. After stirring at room temperature overnight, sodiumbicarbonate (100 mL, 10%) was added, and the organic layer wasseparated. The aqueous layer was extracted twice by DCM (50 mL each).The combined organic layer was dried over magnesium sulfate and thesolvent was removed to obtain product, compound 10a (0.97 g, 90%):

¹H NMR (300 MHz, CD₃OD) δ 8.52 (m, 1), 7.22 (m, 1), 7.01 (m, 1), 3.92(s, 3H); MS (ES+): 135 (M+1).

4-Methoxy-2-cyanopyridine, compound 10a (R⁹=methoxy), (0.97 g, 7.2 mmol)was dissolved in HCl (6N, 50 mL), and refluxed overnight. The HCl wasevaporated and the resulting product was crystallized from acetonile, togive compound 10b (R⁹=methoxy) (0.6 g, 60%).

¹H NMR (300 MHz, CD₃OD) δ 8.65 (m, 1), 7.99 (m, 1), 7.68 (m, 1), 4.21(s, 3H). MS (ES⁻): 152 (M−1).

To 7-methyl α-thiolincosaminide, compound 2b (R¹=Me, R²=Me), (90 mg,0.35 mmol) in DMF (2 mL), TEA (72 mg, 0.7 mmol), BSTFA (276 mg, 1.05mmol) were added at 0° C. and left stirred at room temperature for 1.5hr. Then compound 10b (R⁹=methoxy) (109 mg, 0.7 mmol) and HATU (165 mg,0.53 mmol) were added to the reaction mixture, and stirred at roomtemperature overnight. The DMF was completely removed and the residuewas taken up in EtOAc (50 mL), washed with sodium bicarbonate (10%, 30mL), brine (30 mL), and dried over magnesium sulfate. The solvent wasremoved to obtain a brown oil-like liquid, which was dissolved inmethanol (10 mL) and treated with resin for 1 hr. The resin wasfiltered, and the solvent was removed to obtain the crude material.Purification was carried out on silica gel column chromatography usingEtOAc as eluent to obtain compound 11b (R¹=Me, R²=Me R³=H, R⁹=methoxy)(100 mg, 72%).

¹H NMR (300 MHz, CD₃OD) δ 8.42 (m, 1), 7.64 (m, 1), 7.07 (m, 1), 5.25(d, J=5.4, 1), 4.07–4.87 (m, 3), 3.94 (m, 4), 3.56 (m, 1), 2.99 (m, 2),2.80 (m, 1), 2.22 (m, 1), 2.11 (s, 3), 0.96 (m, 3). MS (ES⁺): 387 (M+1).

To compound 11b (R¹=Me, R²=Me R³=H, R⁹=methoxy) (100 mg, 0.26 mmol) inwater (10 mL), AcOH (3 mL) and MeOH (2 mL), PtO₂ (100 mg) were added andhydrogenated at 55 psi overnight. The solvent was removed to obtain thecrude product. Purification of the crude product was carried out bysilica gel column chromatography using 20% MeOH in DCM to obtain thetitle compound. (9 mg, 9%).

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.17 (dd, J=9.9; 3.3, 1),4.07 (m, 2), 3.79 (m, 1), 3.52 (dd, J=10.5; 3.3, 1), 3.35 (s, 3), 3.18(m, 2), 2.72 (m, 1), 2.16 (m, 1), 2.12 (s, 3) 1.99 (m, 2), 1.50 (m, 1),1.24 (m, 2), 0.90 (d, J=6.9, 6); MS (ES⁺): 393 (M+1).

Example 27 Preparation of 4-(1-ethyl-propyl)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Isopentylpyridine-2-carboxylic acid, compound 10b (R⁹=1-ethyl-propyl),was made by employing general Method P. To 4-(1-ethyl-propyl)-pyridine(8.5 g, 57 mmol) in acetic acid (30 mL), hydrogen peroxide (17.8 g, 30%,57 mmol) was added. The resulting reaction mixture was refluxedovernight. The residue obtained on removal of solvent was dissolved inDCM (100 mL), dried over MgSO₄. After filtering, the solvent was removedto obtain a brown liquid, 4-(1-ethyl-propyl)-pyridine-N-oxide (9 g,95%).

To a solution of trimethylsilyl cyanide (6.5 g, 65 mmol) and4-(1-ethyl-propyl)-pyridine-N-oxide (9 g, 54 mmol) in DCM (25 mL) wasadded a solution of dimethylcarbamoyl chloride (7 g, 65 mmol) in DCM (10mL), dropwise. After stirring at room temperature overnight, sodiumbicarbonate (100 mL, 10%) was added, and the organic layer wasseparated. The aqueous layer was extracted twice with DCM (50 mL). Thecombined organic layer was dried over magnesium sulfate and the solventwas removed to obtain the product, compound 10a (R⁹=1-ethyl-propyl) (9.6g, 100%).

¹H NMR (300 MHz, CD₃OD) δ 8.58 (m, 1), 7.46 (m, 1), 7.26 (m, 1), 2.42(m, 1), 1.77 (m, 4)), 0.78 (t, J=7.5, 6). MS (ES+): 175(M+1).

Compound 10a (R⁹=1-ethyl-propyl) (9.5 g, 54 mmol) was dissolved in HCl(6N, 50 mL) and refluxed overnight. HCl was evaporated and the resultingproduct, compound 10b (R⁹=1-ethyl-propyl), was crystallized fromacetonitrile (10 g, 100%).

¹H NMR (300 MHz, CD₃OD) δ 8.86 (m, 1), 8.45 (m, 1), 8.20 (m, 1), 2.92(m, 1), 1.87 (m, 4), 0.84 (t, J=7.5, 6). MS (ES⁻): 192 (M−1).

To the acid 10b (77 mg, 0.4 mmol) in DMF (2 mL), 7-methylα-thiolincosaminide, compound 2b (R¹=Me, R²=Me), (100 mg, 0.4 mmol) wasadded, followed by HBTU (166 mg, 0.44 mmol) and DIEA (205 mg, 0.8 mmol).The mixture was stirred at room temperature for 2 hr. The product wasobtained on removal of DMF and purified by silica gel columnchromatography using ethyl acetate to provide compound 11b (R¹=Me, R²=MeR³=H, R⁹=1-ethyl-propyl) (150 mg, 89%).

¹H NMR (300 MHz, CD₃OD) δ 8.42 (d, J=5.1, 1), 7.37 (s, 1), 7.32 (m, 1),5.27 (d, J=4.8, 1), 4.21–4.88 (m, 3), 3.85 (d, J=3.6, 1), 3.56 (dd,J=3.3, 10.2, 1), 2.48 (m, 1), 2.11 (m, 4), 1.00 (m, 12). MS (ES+): 427(M+1).

To compound 11b (R¹=Me, R²=Me R³=H, R⁹=1-ethyl-propyl) (130 mg, 0.3mmol) in water (10 mL), AcOH (2 mL) and MeOH (2 mL), PtO₂ (150 mg) wasadded and hydrogenated at 55 psi overnight. The solvent was removed toobtain the crude product. Purification was carried by silica gel columnchromatography using 20% MeOH in DCM to obtain the title compound (40mg, 30%).

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.17 (dd, J=9.9; 3.3, 1),4.10 (m, 2), 3.78 (m, 1), 3.51 (m, 2), 2.81 (m, 2), 2.16 (m, 1), 2.10(s, 3) 1.90 (m, 2), 1.76 (m, 3), 1.40 (m, 8), 0.91 (m, 9); MS (ES+): 433(M+1).

Example 28 Preparation of 4-Isopropyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Isopropylpyridine-2-carboxylic acid, compound (10b) (R⁹=isopropyl),was made by employing general Method P. To 4-isopropylpyridine (5 g,0.041 mol) in acetic acid (60 mL), hydrogen peroxide (30%, 4.7 g, 0.13mol) was added and refluxed over night. After removing the solvent, theresidue was dissolved in DCM dried over magnesium sulfate and taken assuch for the next step. To the resulting compound in dichloromethane (10mL) trimethylsilyl cyanide (7.0 mL, 0.07 mol) and dimethylcarbamylchloride (5.6 mL, 0.05 mol) were added and stirred at room temperaturefor 24 hours. Aqueous potassium carbonate (10%, 50 mL) was added andextracted with dichloromethane (100 mL). The crude product obtained onremoval of solvent was taken up in hydrochloric acid (6N, 30 mL) andrefluxed for 24 hours. Removal of acid followed by crystallization ofthe crude product from acetonitrile resulted in acid 10b (R⁹=isopropyl)(5 g, 75%).

¹H NMR (300 MHz, CD₃OD): δ 8.78 (d, J=6, 1), 8.42 (s, 1), 8.16 (d,J=6.0, 1), 3.25 (m, 1), 1.33 (d, J=9.0, 6) MS (ES⁻): 164 (M−1).

To the amine, compound 2b (R¹=Me, R²=Me), (140 mg, 0.56 mmol) in DMF (3mL), BSTFA (0.59 mL, 2.24 mmol) and triethylamine (0.18 mL, 1.26 mmol)were added at 0° C. and the reaction mixture was stirred at roomtemperature for 3 hours. Acid 10b (R⁹=isopropyl) (188 mg, 1.13 mmol) andHATU (319 mg, 0.84 mmol) were combined and left stirred for further 4hours at room temperature. The DMF was removed and the residue wasextracted with ethyl acetate (100 mL) and washed with saturatedbicarbonate (40 mL). The product obtained on removal of solvent wastaken up in methanol and treated with Dowex™ H⁺ resin for 1 hour. Afterfiltering the resin, methanol was removed to obtain the crude product.It was then purified on silica gel column using 10% methanol indichloromethane as eluent to provide compound 11b (R¹=Me, R²=Me R³=H,R⁹=isopropyl) (120 mg, 53%).

¹H NMR (300 MHz, CD₃OD): δ 8.42 (d, J=5.1, 1), 7.37 (s, 1), 7.32 (m, 1),5.27 (d, J=4.8,1), 4.21–4.88 (m, 3), 3.85 (d, J=3.6, 1), 3.56 (dd,J=3.3, 10.2, 1), 2.48 (m, 1), 2.11 (m, 1), 2.10 (s, 3), 1.20 (m, 12). MS(ES+): 399 (M+1).

To 11b (R¹=Me, R²=Me R³=H, R⁹=isopropyl) (100 mg, 0.257 mmol) in meth (5mL), water (10 mL) and acetic acid (5 mL), platinum dioxide (100 mg,0.44 mmol) was added and hydrogenated at 60 psi for 16 h. Afterfiltering the catalyst, the solvent was stripped off to obtain the crudeproduct which was then purified on silica gel column chromatographyusing 10% methanol in dichloromethane as eluent. The lower R_(f)compound was the title compound (10 mg, 9%).

¹H NMR (300 MHz, CD₃OD): δ 5.24 (d, J=5.7, 1), 4.17 (dd, J=9.9; 3.3, 1),4.10 (m, 2), 3.80 (m, 1), 3.51 (m, 1), 3.16 (m, 1), 2.61 (m, 1), 2.16(m, 1), 2.10 (s, 3) 1.90 (m, 1), 1.76 (m, 1), 1.50–1.09 (m, 5), 0.91 (m,12); MS (ES+): 405 (M+1).

Example 29 Preparation of 4-Butyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

4-Butylpyridine was prepared by adding potassium-t-butoxide (0.68 g, 6mmol) to propylphosphonium bromide (Aldrich) (2.4 g, 6.0 mmol) in THF(10 mL), at 0° C. and stirring at room temperature for 1 hour.Pyridine-4-carbaldehyde (428 mg, 4 mmol) was added and the reactionmixture stirred for 2 h. The reaction mixture was then poured into waterand extracted with ethyl acetate. The product obtained after removingthe solvent was taken as such in methanol (30 mL) to which palladium oncarbon (10%, 300 mg) was added and hydrogenated at 1 atm pressure overnight. Removal of solvent and purification on column chromatographyusing ethyl acetate resulted in pure product 4-butylpyridine (500 mg,92%):

¹H NMR (CDCl₃): δ 8.42 (d, J=6.0, 2), 7.05 (d, J=6.0, 1), 2.60 (t,J=6.5, 2), 1.62 (m, 2), 1.37 (m, 2), 0.93 (t, J=7.0, 3). MS (ES+): 136(M+1).

4-Butylpyridine-2-carboxylic acid, compound (10b) (R⁹=butyl), was madeemploying general Method P. To 4-butylpyridine (2 g, 0.014 mol) inacetic acid (15 mL), hydrogen peroxide (30%, 5 mL, 0.056 mol) was addedand refluxed over night. After removing the solvent, the residue wasdissolved in DCM dried over magnesium sulfate and taken as such for thenext step. To the compound from the previous step in dichloromethane (10mL) trimethylsilyl cyanide (3.92 mL, 0.029 mol) and dimethylcarbamoylchloride (2.67 mL, 0.028 mol) was added and stirred at room temperaturefor 24 hours. Aqueous potassium carbonate (10%, 50 mL) was added andextracted with dichloromethane (100 mL). The crude product obtained onremoval of solvent was taken up in hydrochloric acid (6N, 30 mL) andrefluxed for 24 hours. Removal of acid followed by crystallization ofthe crude product from acetonitrile resulted in acid 10b (R⁹=butyl) (1.5g, 60%).

¹H NMR (CDCl₃): δ 8.92 (d, J=6.0, 1), 8.65 (s, 1), 8.27 (m, 1), 3.23 (t,J=6.5, 2), 1.98 (m, 2), 1.67 (m, 2), 1.20 (t, J=7.0, 3). MS (ES−): 178(M−1).

To the amine, compound 2b (R¹=Me, R2=Me), (140 mg, 0.56 mmol) in DMF (3mL), BSTFA (0.59 mL, 2.24 mmol) and triethylamine (0.18 mL, 1.26 mmol)were added at 0° C. and then stirred at room temperature for 3 hours.Acid 10b (R⁹=butyl) (203 mg, 1.13 mmol) and HATU (319 mg, 0.84 mmol)were added and the reaction mixture was stirred for 4 more hours at roomtemperature. The DMF was removed and the residue was extracted withethyl acetate (100 mL) and washed with saturated bicarbonate (40 mL).The product obtained on removal of solvent was taken up in methanol andtreated with Dowex™ H⁺ resin for 1 hour. After filtering the resin,methanol was removed to obtain the crude product. The product was thenpurified on silica gel column using ethyl acetate as eluent to providefor compound 11b (R¹=Me, R²=Me R³=H, R⁹=butyl) (200 mg, 86%).

¹H NMR (CDCl₃) δ 8.40 (d, J=4.2, 1), 8.01 (s, 1), 7.29 (m, 1), 5.40 (d,J=5.4, 1), 4.02–4.36 (m, 3), 4.80 (s, 1), 3.48–3.60 (m, 1), 3.72 (t,J=6.0, 2), 2.49 (m, 1), 2.20 9 s, 3), 1.67 (m, 4), 1.40 (m, 3),0.98–1.18 (m, 9). Mass 413 (M+1).

To compound 11b (R¹=Me, R²=Me R³=H, R⁹=butyl) (200 mg, 0.49 mmol) inmethanol (5 mL), water (10 mL) and acetic acid (5 mL), platinum dioxide(100 mg, 0.44 mmol) was added and hydrogenated at 60 psi for 16 h. Afterfiltering the catalyst, the solvent was stripped off to obtain the crudeproduct, which was then purified on silica gel column chromatographyusing 20% methanol in dichloromethane as eluent. The lower R_(f)fractions provided the title compound (60 mg, 29%).

¹H NMR (CDCl₃): δ 5.20 (d, J=3.6,1), 4.20 (dd, J=3.0, 4.8, 1), 4.04 (m,2), 3.80 (d, J=3.0, 1), 3.61–3.66 (m, 1), 3.52 (dd, J=3.3, 10.2), 2.88(m, 1), 2.17 (m, 1), 2.14 (s, 3), 1.87 (m, 2), 1.62 (m, 2), 1.32 (m, 6),0.89 (m, 9); MS (ES+): 419 (M+1).

Example 30 Preparation of 4-Cyclohexyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

4-phenylpyridine-2-carboxylic acid, compound 10b (R⁹=phenyl), was madeby employing general Method P. To 4-phenylpyridine-N-oxide (1 g, 5.84mmol) in dichloromethane (10 mL) trimethylsilyl cyanide (1.5 mL, 11.6mmol) and dimethylcarbamoyl chloride (1 mL, 11.6 mmol) were added andthe reaction mixture was stirred at room temperature for 24 hours. Anaqueous potassium carbonate solution (10%, 10 mL) was added andextracted with dichloromethane (100 mL). The crude product obtained onremoval of solvent was taken up in hydrochloric acid (6N, 30 mL) andrefluxed for 24 hours. Removal of acid followed by crystallization ofthe crude product from acetonitrile resulted in acid 10b (R^(9′)=phenyl)(1 g, 86%).

MS (ES⁻): 198 (M−1); ¹H NMR (300 MHz, CD₃OD) δ 7.64–7.67 (m, 3),8.02–8.06 (m, 2), 8.53–8.56 (m, 1), 8.82 (s, 1), 8.82–8.90 (m, 1).

To the amine 2b (R¹=Me, R²=Me) (102 mg, 0.42 mmol) in DMF (5 mL), BSTFA(0.549 mL, 2.1 mmol) and triethylamine (0.183 mL, 1.26 mmol) was addedat 0° C. and then stirred at room temperature for 3 hours. Acid 10b(R⁹=phenyl) (158 mg, 0.80 mmol) and HATU (302 mg, 0.80 mmol) were addedand the reaction was stirred for an additional 4 hours at roomtemperature. The DMF was removed and the residue was extracted withethyl acetate (100 mL) and washed with saturated bicarbonate (40 mL).The product obtained on removal of solvent was taken up in methanol andtreated with Dowex™ H⁺ resin for 1 hour. After filtering the resin,methanol was removed to obtain the crude product. The resulting residuewas then purified by silica gel chromatography using 10% methanol indichloromethane as eluent to provide compound 11b (R¹=Me, R²=Me R³=H,R^(9′)=phenyl) (50 mg, 58%).

TLC: Rf=0.70 (10% MeOH/DCM); MS (ES⁺): 435 (M+1); ¹H NMR (300 MHz,CDCl₃) δ 1.01 (t, J=6.6, 6), 2.12 (s, 3), 2.28 (m, 1), 3.56 (dd, J=3.3,10.5, 1), 3.90 (d, J=3.3, 1), 4.12 (dd, J=5.4, 10.5, 1), 4.27–4.36 (m,2), 4.52 (m, 2), 5.26 (d, J=5.7, 1), 7.48–7.55 (m, 3), 7.77–7.80 (m, 2),7.83–7.85 (m, 1), 8.37 (s, 1), 8.69 (d, J=5.4, 1).

To compound 11b (R¹=Me, R²=Me R³=H, R^(9′)=phenyl) (40 mg, 0.09 mmol) inmethanol (5 mL), water (10 mL) and acetic acid (5 mL), platinum dioxide(100 mg, 0.44 mmol) was added and the reaction mixture shaken at 60 psihydrogen for 16 h. The catalyst was removed by filtration, and thesolvent was evaporated to obtain the crude product, which was thenpurified on silica gel column chromatography using 10% methanol indichloromethane to provide the title compound (10 mg, 25%).

TLC: R_(f)=0.22 (20% MeOH/DCM); MS (ES⁺): 447 (M+1); ¹H NMR (300 MHz,CD₃OD) δ 0.90 (d, J=6.8, 6), 0.93–1.05 (m, 5), 1.20 (m, 6), 1.33–1.47(m, 4), 1.75 (m, 6), 2.10 (s, 3), 2.18–2.22 (m, 1), 2.97 (t, J=12.3, 1),3.39–3.52 (m, 2), 3.70–3.78 (m, 2), 4.05–4.21 (m, 3), 5.23 (d, J=5.7,1).

Example 31 Preparation of4-Ethyl-1-(2-hydroxy-ethyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide;

To the product of Example 1 (28 mg, 0.07 mmol) in methanol (2 mL),ethylene oxide (0.5 mL) was added and stirred at 4° C. overnight. Thesolvent was removed and the resulting product was purified by columnchromatography using 20% MeOH in DCM as eluent to obtain the titlecompound (16 mg, 51%) as a white powder.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=6, 1), 4.27 (m, 1), 4.10 (m, 2),3.95 (m, 1), 3.79–3.50 (m, 4), 3.85 (m, 1) 3.74 (m, 1), 3.26 (m, 1),2.91 (m, 2), 2.33 (m, 1), 2.13 (m, 4), 1.92 (m, 1), 1.71 (m, 1), 1.17(m, 7), 0.94 (m, 9); MS (ES⁺): 435 (M+1).

Example 32 Preparation of1-(2-Hydroxy-ethyl)-4-pentyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was prepared using the procedures of Example 32 withthe product from Example 25 as the starting material.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=6.0, 1), 4.19 (m, 3), 3.79 (d,J=3.3, 1), 3.74 (m, 1), 3.65 (m, 1), 3.54 (dd, J=3.0, 10.2, 1), 3.25 (m,2), 2.82 (m, 2), 2.14 (m, 4), 1.89 (m, 1), 1.72 (m, 1), 1.28 (m, 12),0.94 (m, 9); MS (ES⁺): 477 (M+1).

Example 33 Preparation of1-(2-Hydroxy-ethyl)-4-propyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the procedures of Example 32 with theproduct of Example 17 as the starting material.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=6.0, 1), 4.19 (dd, J=9.6; 3.3, 1),4.11 (m, 2), 3.79 (d, J=3.3, 1), 3.75 (m, 1), 3.65 (m, 1), 3.54 (m, 1),3.28 (m, 1), 2.82 (m, 2), 2.27 (m, 5) 1.90 (m, 1), 1.71 (m, 1), 1.36 (m,8), 0.94 (m, 9); MS (ES+): 449 (M+1).

Example 34 Preparation of2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylic acid9H-fluoren-9-ylmethyl ester

To the product of Example 17 (50 mg, 0.123 mmol) in water (3 mL) anddioxane (3 mL), Fmoc-Cl (38 mg, 0.197 mmol) and sodium carbonate (25 mg,0.246 mmol) were added and the reaction mixture was stirred over nightat room temperature. The solvents were removed and the crude materialwas loaded into a silica gel column and eluted with ethyl acetate toobtain the title compound as a white solid (30 mg, 51%).

TLC: Rf=0.5 (EtOAc). MS (ES⁺)=627 (M+1), 649 (M+Na); ¹H NMR (CD₃OD, 200MHz): 7.79 (d, J=4.6 Hz, 2), 7.59–7.62 (m, 2), 7.28–7.41 (m, 4), 5.19(d, J=3.8 Hz, 1), 4.45 (m, 2), 4.24 (t, J=4.2, 1), 3.99–4.15 (m, 4),3.93 (m, 1), 3.47–3.50 (m, 2), 2.05 (s, 3), 1.87 (m, 1), 1.67 (s, 2),1.50 (m, 1), 1.30 (m, 4), 0.86–0.91 (m, 9).

Example 35 Preparation of2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylic acid ethyl ester

To the product of Example 17 (50 mg, 0.123 mmol) in water (3 mL) anddioxane (3 mL), ethyl chloroformate (20 mg, 0.147 mmol) and sodiumcarbonate (25 mg, 0.246 mmol) were added and stirred over night at roomtemperature. The solvents were removed and the crude material was loadedinto a silica gel column and eluted with ethyl acetate to obtain thetitle compound as a white solid (40 mg, 52%).

TLC: Rf=0.28 (EtOAc). MS (ES⁺)=477 (M+1), 499 (M+Na); ¹H NMR (CD₃OD, 200MHz): 5.22 (d, J=3.6 Hz, 1), 4.27 (m, 1), 4.03–4.14 (m, 5), 3.96 (bs,1), 3.62 (m, 1), 3.54 (d, J=2.2 Hz, 1), 3.52 (d, J=2.2 Hz, 1), 2.08 (s,3), 1.93–2.03 (m, 2), 1.75–1.85 (m, 3), 1.61 (m, 2), 1.33 (m, 4),1.22–1.28 (m, 3), 0.90–0.94 (m, 9).

Example 36 Preparation of2-[2-Methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propylcarbamoyl]-4-propyl-piperidine-1-carboxylic acid phenyl ester;

To the product of Example 17 (50 mg, 0.123 mmol) in water (3 mL) anddioxane (3 mL), phenyl chloroformate (40 mg, 0.246 mmol) and sodiumcarbonate (25 mg, 0.246 mmol) were added and the reaction mixture wasstirred over night at room temperature. The solvents were removed andthe crude material was loaded into a silica gel column and eluted withethyl acetate to obtain the title compound as a white solid (30 mg,47%).

TLC: Rf=0.4 (EtOAc). MS (ES⁺)=526 (M+1), 548 (M+Na); ¹H NMR (CD₃OD, 200MHz): 7.36 (t, J=3.8 Hz, 2), 7.17–7.23 (m, 10), 7.10 (d, J=3.6 Hz, 2),5.20 (d, J=3.6 Hz, 1), 4.09 (m, 3), 3.93 (d, J=2.2 Hz, 1), 3.82 (m, 2),3.46 (m, 2), 2.01. (s, 3), 2.00 (m, 1), 1.71 (m, 1), 1.46–1.36 (m, 4),0.96–0.90 (m, 9).

Example 37 Preparation of4-(4,4-Difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of aldehyde 8a, prepared by first step in general MethodL, (510 mg, 1.47 mmol, 1 equiv) in benzene (8 mL) was added1-triphenylphosphoranylidene-2-propanone (Aldrich) (702 mg, 2.2 mmol,1.5 equiv). The reaction mixture was refluxed overnight and the solventwas removed under vacuum. The residue was purified by chromatography togive 4-(4-oxo-pent-2-enyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester as an oil (237 mg, 42%).

MS (ESPOS): 410.2 [M+Na]⁺, 288.3 [M−Boc+H]^(+;)MS (ESNEG): 386.2 [M−H]⁻.

To a solution of 4-(4-oxo-pent-2-enyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (193 mg, 0.5 mmol, 1 equiv) in benzene(0.9 mL) was added a solution of triphenylphosphine-copper (I) hydridehexamer in benzene (3.6 mL). The mixture was stirred at rt overnight andhexane (13 mL) was added. The mixture was filtered and the filtrate wasevaporated. The residue was purified by chromatography to give4-(4-oxo-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (127 mg, 65%).

¹H NMR (300 MHz, CDCl₃) δ 7.35–7.30 (m, 5), 5.25–5.04 (m, 2), 4.42–4.25(m, 1), 3.77–3.62 (m, 1), 3.00–2.85 (m, 1), 2.39 (t, J=7, 2), 2.34–1.47(m, 7), 2.10 (s, 3), 1.43 (s, 3H), 1.31 (s, 6H); MS (ESPOS): 412.3[M+Na]⁺, 290.3 [M−Boc+H]⁺; MS (ESNEG): 388.4 [M−H]⁻.

To a solution of 4-(4-oxo-pentyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (155 mg, 0.40 mmol, 1 equiv) indichloromethane (1.5 mL) at −78° C. was added DAST (0.21 mL, 1.60 mmol,4 equiv). The reaction mixture was warmed to rt and stirred at rt for 3h, followed by additional DAST (0.32 mL, 2.4 mmol, 6 equiv) at −78° C.The mixture was warmed to rt and stirred overnight. Then the mixture wasdiluted with dichloromethane, washed with sat. aqueous NaHCO₃ (1×),dried, and evaporated. The residue was purified by chromatography togive 4-(4,4-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester as a yellow oil (88 mg, 54%).

MS (ESPOS): 434.2 [M+Na]⁺, 312.3 [M−Boc+H]⁺.

To a solution of 4-(4,4-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylicacid 1-tert-butyl ester 2-methyl ester (88 mg, 0.21 mmol, 1 equiv) inTHF (1.2 mL) and water (0.4 mL) was added lithium hydroxide monohydrate(45 mg, 1.07 mmol, 5 equiv). The reaction mixture was stirred at rtovernight. The THF was removed under vacuum. The residue was dilutedwith water and washed with ether. The aqueous layer was taken up inethyl acetate, partitioned with 10% citric acid. The organic layer waswashed with water (1×), brine (1×), dried and concentrated to give4-(4,4-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester (66 mg, 96%).

¹H NMR (300 MHz, CDCl₃) δ 4.39–4.34 (m, 1), 3.57–3.48 (m, 1), 2.92–2.83(m, 1), 2.57–2.50 (m, 1), 2.30–2.18 (m, 1), 1.91–1.73 (m, 3), 1.64–1.36(m, 7), 1.48 (s, 9); MS (ESPOS): 344.3 [M+Na]⁺, 222.3 [M−Boc+H]⁺; MS(ESNEG): 320.2 [M−H]⁻.

To a solution of compound 2b (R¹=Me, R²=Me) (50 mg, 0.20 mmol, 1 equiv)in dry DMF (0.5 mL) at 0° C. was added triethylamine (88.3 up, 0.64mmol, 3.2 equiv), followed by the addition of BSTFA (79.2 μL, 0.30 mmol,1.5 equiv). The reaction mixture was stirred at 0° C. for 10 minutes,and then was stirred at rt for 50 minutes. The reaction mixture wasadded to the acid (66 mg, 0.21 mmol, 1 equiv) in a 25 mL round bottomflask, followed by the addition of HATU (96.8 mg, 0.25 mmol, 1.25equiv). The reaction mixture was stirred at rt for 3 h. The reactionmixture was evaporated to dryness, taken up in ethyl acetate, washedwith 10% citric acid, water, sat. NaHCO₃ and brine. The organic layerwas dried over Na₂SO₄ and evaporated and used in the next step withoutadditional purification.

To a solution of the above Boc protected lincosamide in DCM (9 mL) withmethyl sulfide (0.20 mL) were added trifluoroacetic acid (3 mL) andwater (0.20 mL). The reaction mixture was stirred at rt for 1 h. Thesolvent was removed under vacuum and co-evaporated with toluene twice.The residue was purified by chromatography to provide the title compound(68 mg, 75%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.4, 1), 4.16 (dd, J=3.3, 9.9, 1),4.11–4.00 (m, 3), 3.75 (d, J=3.3, 1), 3.51 (dd, J=3.3, 10.2, 1),3.40–3.32 (m, 1), 2.71 (dd, J=8.2, 10.6, 1), 2.23–2.05 (m, 3), 2.10 (s,3), 1.98–1.76 (m, 3), 1.63–1.39 (m, 7), 0.94–0.87 (m, 6). MS (ESPOS):455.3 [M+H]⁺.

Example 38 Preparation of4-(3,3-Difluoro-butyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

Ethyl triphenylphosphonium bromide (Aldrich) (2.92 g, 7.86 mmol, 3.9equiv) and potassium-t-butoxide (0.61 g, 5.44 mmol, 2.7 equiv) weresuspended in toluene (26 mL) under nitrogen with vigorous stirring.After 4 h, a solution of aldehyde 8a prepared by the first step ingeneral Method L (700 mg, 2.01 mmol, 1 equiv) in toluene (17 mL) wasadded dropwise. The reaction mixture was stirred at rt for 2 h anddiluted with ethyl acetate (150 mL). The organic layer was washed withwater (2×), brine, dried and concentrated. The residue was purified bychromatography to give a clear oil4-but-2-enyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (360 mg, 50%).

MS (ESPOS): 260.3 [M+H−Boc]⁺.

To a solution of 4-but-2-enyl-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (149 mg, 0.42 mmol, 1 equiv) in DMF(1.4 mL) and water (0.2 mL) were added palladium(II)chloride (7.4 mg,0.042 mmol, 0.1 equiv) and copper(I) chloride (41.1 mg, 0.42 mmol, 1equiv). The mixture was stirred at 50° C. overnight with oxygen bubblinginto the mixture. The mixture was filtered and the filtrate wasconcentrated under high vacuum. The residue was diluted with ethylacetate, washed with water (1×), brine (1×), dried and concentrated. Theresidue was purified by preparative TLC to give4-(3-oxo-butyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (110 mg, 71%).

¹H NMR (300 MHz, CDCl₃) δ 7.35–7.28 (m, 5), 5.24–5.03 (m, 2), 4.43–4.25(m, 1), 3.75–3.61 (m, 1), 3.01–2.87 (m, 1), 2.44–2.35 (m, 2), 2.28–2.15(m, 1), 2.11 (s, 3), 2.09–1.98 (m, 1), 1.91–1.51 (m, 3), 1.43 (s, 3.4H),1.31 (s, 5.6H). MS (ESPOS): 398.3 [M+Na]⁺, 276.3 [M−Boc+H]⁺.

To a solution of the 4-(3-oxo-butyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (110 mg, 0.29 mmol, 1 equiv) indichloromethane (1.1 mL) at −78° C. was added DAST (0.16 mL, 1.17 mmol,4 equiv). The reaction mixture was warmed to rt and stirred at rt for 3h, followed by additional DAST (0.23 mL, 1.76 mmol, 6 equiv) at −78° C.The mixture was warmed to rt and stirred overnight. Then the mixture wasdiluted with dichloromethane, washed with sat. aqueous NaHCO₃ (1×),dried, evaporated. The residue was purified by chromatography to give4-(3,3-Difluoro-butyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester 2-methyl ester (92.7 mg, 80%).

MS (ESPOS): 420.3 [M+Na]⁺.

To a mixture of 4-(3,3-difluoro-butyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (92.7 mg, 0.23 mmol, 1 equiv) in THF(1.2 mL) and water (0.4 mL) was added lithium hydroxide monohydrate (49mg, 1.17 mmol, 5 equiv). The reaction mixture was stirred at rtovernight the THF was removed under vacuum. The residue was diluted withwater, washed with ether. The aqueous layer was taken up in ethylacetate, partitioned with 10% citric acid. The organic layer was washedwith water (1×), brine (1×), dried and concentrated to give a whitesolid, 4-(3,3-difluoro-butyl)-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester (59.7 mg, 83%).

¹H NMR (300 MHz, CDCl₃) δ 4.40–4.36 (m, 1), 3.59–3.52 (m, 1), 2.94–2.86(m, 1), 2.55–2.48 (m, 1), 2.33–2.15 (m, 1), 1.92–1.73 (m, 3), 1.66–1.40(m, 5), 1.47 (s, 9); MS (ESPOS): 330.2 [M+Na]⁺, 208.2 [M−Boc+H]⁺; MS(ESNEG): 306.1 [M−H]⁻.

To a solution of compound 2b (R¹=Me, R²=Me) (50 mg, 0.20 mmol, 1 equiv)in dry DMF (0.5 mL) at 0° C. was added triethylamine (88.3 up, 0.64mmol, 3.2 equiv), followed by the addition of BSTFA (79.2 μL, 0.30 mmol,1.5 equiv). The reaction mixture was stirred at 0° C. for 10 minutes,and then was stirred at rt for 50 minutes. The reaction mixture wasadded to the acid 4-(3,3-difluoro-butyl)-pyrrolidine-1,2-dicarboxylicacid 1-tert-butyl ester (59.7 mg, 0.20 mmol, 1 equiv) in a 25 mL roundbottom flask, followed by the addition of HATU (93.3 mg, 0.25 mmol, 1.25equiv). The reaction mixture was stirred at rt for 3 h. The reactionmixture was evaporated to dryness, taken up in ethyl acetate, washedwith 10% citric acid, water, sat. NaHCO₃ and brine. The organic layerwas dried over Na₂SO₄ and evaporated to give a syrup.

To a solution of the above syrup in DCM (9 mL) with methyl sulfide (0.20mL) were added trifluoroacetic acid (3 mL) and water (0.20 mL). Thereaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (63 mg, 72%) asa white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.22–4.13 (m, 2),4.10–4.04 (m, 2), 3.76 (d, J=2.4, 1), 3.54–3.42 (m, 2), 2.84–2.76 (m,1), 2.29–1.83 (m, 5), 2.10 (s, 3), 1.67–1.51 (m, 6), 0.95–0.87 (m, 6).MS (ESPOS): 441.3 [M+H]⁺

Example 39 Preparation of4-(3,3-Difluoro-pentyl)-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of compound 7c (R^(9′)=2-penteneyl) prepared using methodsof general Method K (323.7 mg, 0.87 mmol, 1. equiv) in DMF (2.8 mL) andwater (0.4 mL) at 0° C. were added palladium (II) chloride (15.4 mg,0.087 mmol, 0.1 equiv) and copper(I) chloride (85.9 mg, 0.87 mmol, 1equiv). The mixture was stirred at 50° C. overnight with oxygen bubblinginto the mixture. The mixture was filtered and the filtrate wasconcentrated under high vacuum. The residue was diluted with ethylacetate, washed with water (1×), brine (1×), dried and concentrated. Theresidue was purified by preparative TLC to provide4-(3-oxo-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (242 mg, 72%).

MS (ESPOS): 412.3 [M+Na]⁺, 290.3 [M−Boc+H]⁺.

To 4-(3-oxo-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (242 mg, 0.62 mmol, 1 equiv) in dichloromethane (2.3 mL)at −78° C. was added DAST (0.33 mL, 2.49 mmol, 4 equiv). The reactionmixture was warmed to rt and stirred at rt for 3 h, followed by anaddition of more DAST (0.49 mL, 3.73 mmol, 6 equiv) at −78° C. Themixture was warmed to rt and stirred overnight. Then the mixture wasdiluted with dichloromethane, washed with sat. aqueous NaHCO₃ (1×),dried, evaporated. The residue was purified by chromatography to4-(3,3-Difluoro-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester 2-methyl ester (117 mg, 46%).

¹H NMR (300 MHz, CDCl₃ ) δ 7.35–7.26 (m, 5), 5.25–5.04 (m, 2), 4.44–4.27(m, 1), 3.79–3.64 (m, 1), 3.02–2.89 (m, 1), 2.32–2.17 (m, 1), 2.13–2.02(m, 1), 1.91–1.68 (m, 5), 1.57–1.47 (m, 2), 1.44 (s, 3.5H), 1.31 (s,5.5H), 0.97 (t, J=7.5, 3).

MS (ESPOS): 434.3 [M+Na]⁺, 312.3 [M−Boc+H]⁺.

To a solution of 4-(3,3-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylicacid 1-tert-butyl ester 2-methyl ester (106 mg, 0.26 mmol, 1 equiv) inTHF (2.4 mL) and water (0.8 mL) was added lithium hydroxide monohydrate(54 mg, 1.29 mmol, 5 equiv). The reaction mixture was stirred at rtovernight. THF was removed under vacuum. The residue was diluted withwater (10 mL), washed with ether (20 mL). The aqueous layer was taken upin ethyl acetate (50 mL), partitioned with 10% citric acid (25 mL). Theorganic layer was washed with water (1×), brine (1×), dried andconcentrated to give4-(3,3-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester as a clear oil (82.1 mg, 99%).

¹H NMR (300 MHz, CDCl₃) δ 4.40–4.36 (m, 1), 3.58–3.51 (m, 1), 2.94–2.86(m, 1), 2.57–2.51 (m, 1), 2.30–2.15 (m, 1), 1.92–1.72 (m, 5), 1.62–1.53.(m, 2), 1.48 (s, 9), 0.99 (t, J=7.5, 3); MS (ESPOS): 344.3 [M+Na]⁺,222.3 [M−Boc+H]⁺.

To a solution of compound 2b (R¹=Me, R²=Me) (50 mg, 0.20 mmol, 1 equiv)in dry DMF (0.5 mL) at 0° C. was added triethylamine (88.3 uL,0.64,mmol, 3.2 equiv), followed by the addition of BSTFA (79.2 uL, 0.30mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for 10minutes, and then was stirred at rt for 50 minutes. The reaction mixturewas added to the acid4-(3,3-difluoro-pentyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester (76.6 mg, 0.24 mmol, 1.2 equiv) in a 25 mL round bottom flask,followed by the addition of HATU (111.9 mg, 0.29 mmol, 1.5 equiv). Thereaction mixture was stirred at rt for 3 h. The reaction mixture wasevaporated to dryness, taken up in ethyl acetate (60 mL), washed with10% citric acid (30 mL), water (30 mL), sat. NaHCO₃ (30 mL) and brine.The organic layer was dried over Na₂SO₄ and evaporated to give a yellowoil.

To a solution of the above oil in DCM (9 mL) with methyl sulfide (0.20mL) were added trifluoroacetic acid (3 mL) and water (0.20 mL). Thereaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (72 mg, 80%) asa white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.20–4.04 (m, 4), 3.76 (d,J=2.7, 1), 3.51 (dd, J=3.4, 10.3, 1), 3.43 (dd, J=6.9, 10.8,1), 2.77(dd, J=8.4, 10.8, 1), 2.30–2.05 (m, 3), 2.10 (s, 3), 2.03–1.76 (m, 5),1.64–1.54 (m, 2), 1.03–0.89 (m, 9); MS (ESPOS): 455.4 [M+H]⁺.

Example 40 Preparation of4-(3,3-Difluoro-pentyl)-1-(2-hydroxy-ethyl)-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of the title compound of Example 39 (17.9 mg, 0.039 mmol)in MeOH (2 mL) at 0° C. was added ethylene oxide (0.4 mL). The reactionmixture was stirred at 4° C. overnight. The reaction mixture wasconcentrated and purified by chromatography to give the title compoundas a white solid (8.2 mg, 42%).

¹H NMR (300 MHz, CD₃OD) δ 5.23 (d, J=5.7, 1), 4.13–4.05 (m, 3), 3.75 (d,J=3.6, 1), 3.72–3.57 (m, 2), 3.53 (dd, J=3.3, 10.2, 1), 3.41–3.36 (m,1), 3.22 (dd, J=3.3, 10.8, 1), 2.88–2.78 (m, 1), 2.63–2.54 (m, 1),2.18–1.99 (m, 4), 2.10 (s, 3), 1.93–1.75 (m, 5), 1.57–1.46 (m, 2),1.01–0.90 (m, 9); MS (ESPOS): 499.6 [M+H]⁺; MS (ESNEG): 497.5 [M−H]⁻.

Example 41 Preparation of4-(3,3-Difluoro-propyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

Compound 14c (R⁹=3,3-difluoro-propyl) is prepared using the methodsdescribed in general Method R.

To a dry flask were added compound 14a (1.4 g, 5.32 mmol, 1 equiv),triphenylphosphine (111.6 mg, 0.43 mmol, 0.08 equiv), copper (I) iodide(81 mg, 0.43 mmol, 0.08 equiv), palladium acetate (47.7 mg, 0.21 mmol,0.04 equiv) and triethylamine (20 mL). The mixture was deaerated withnitrogen, followed by addition of propiolaldehyde diethyl acetyl (1.36g, 10.65 mmol, 2 equiv). The mixture was stirred at rt for 3 hrs. Thesolvent was removed under vacuum to give a dark residue. The residue waspurified by chromatography to give a yellow oil, compound 14b(R^(9′)=3,3-Diethoxy-prop-1-ynyl) (1.4 g, 100%).

¹H NMR (300 MHz, CDCl₃) δ 8.69 (dd, J=0.8, 5.0, 1), 8.15 (dd, J=0.8,1.4, 1), 7.49 (dd, J=1.7, 5.0, 1), 5.48 (s, 1), 3.99 (s, 3), 3.82–3.73(m, 2), 3.71–3.62 (m, 2), 1.26 (t, J=7.2, 6). MS (ESPOS): 264.5 [M+H]⁺.

To a solution of 14b (R^(9′)=3,3-Diethoxy-prop-1-ynyl) (1.4 g, 5.32mmol) in methanol (100 mL) was added 10% palladium on carbon (0.3 g).The mixture was purged and charged with hydrogen (1 atm) and shaken atrt overnight. The palladium was removed by filtration and the filtratewas concentrated to give 14c (R⁹=3,3-diethoxy propyl) as an oil (1.39 g,98%).

¹H NMR (300 MHz, CDCl₃) δ 8.60 (d, J=5.1, 1), 7.98 (d, J=0.9, 1),7.31–7.28 (m, 1), 4.45 (t, J=5.4, 1), 3.98 (s, 3), 3.72–3.58 (m, 2),3.52–3.39(m, 2), 2.79–2.72 (m, 2), 1.99–1.90 (m, 2), 1.22–1.15 (m, 6).

To a mixture of 14c (R⁹=3,3-diethoxy propyl) (0.68 g, 2.55 mmol) inacetic acid (8 mL) and water (2 mL) was added conc. hydrochloric acid (2drops). The mixture was stirred at rt overnight and the solvent wasremoved under high vacuum. The residue was diluted with ethyl acetate,washed with sat. sodium bicarbonate (1×), brine (1×). The organic layerwas dried and concentrated to give4-(3-Oxo-propyl)-pyridine-2-carboxylic acid methyl ester as a yellow oil(0.27 g, 55%).

To a solution of aldehyde 4-(3-oxo-propyl)-pyridine-2-carboxylic acidmethyl ester (0.27 g, 1.4 mmol, 1 equiv) in DCM (5 mL) at −78° C. wasadded DAST (0.91 g, 5.6 mmol, 4 equiv). The mixture was warmed to rt andstirred overnight. The mixture was diluted with dichloromethane (60 mL),washed with sat. aqueous NaHCO₃ (1×), dried, and evaporated. The residuewas purified by prep.

TLC (5% MeOH in DCM) to 4-(3,3-difluoro-propyl)-pyridine-2-carboxylicacid methyl ester (137 mg, 45%): ¹H NMR (300 MHz, CDCl₃) δ 8.64 (d,J=5.1, 1), 8.00–7.98 (m, 1), 7.33–7.29 (m, 1), 5.85 (dddd, J=4.1, 4.1,56.4, 56.4, 1), 3.99 (s, 3), 2.90–2.83 (m, 2), 2.28–2.09 (m, 2); MS(ESPOS): 216.4 [M+H]⁺.

To a solution of 4-(3,3-difluoro-propyl)-pyridine-2-carboxylic acidmethyl ester (130 mg, 0.6 mmol) (compound 14c R⁹=3,3-difluoro-propyl) inMeOH (3 mL) and water (3 mL) were added conc. HCl (0.25 mL, 3.0 mmol, 5equiv) and platinum oxide (65 mg). The mixture was purged and chargedwith hydrogen (1 atm) and stirred overnight. The platinum oxide wasremoved by filtration and the filtrate was evaporated to give a clearsyrup. To the above residue were added 2N NaOH (1.21 mL) and t-butylalcohol (0.7 mL). The mixture was stirred at rt for 2 hrs. Thendi-t-butyl dicarbonate (0.16 g, 0.73 mmol) was added. The mixture wasstirred at rt overnight. The solvent was removed under vacuum. Theresidue was diluted with water (10 mL), was washed with ether (20 mL).The aqueous layer was acidified with 2N HCl to pH=2.0, and extractedwith ethyl acetate (2×). The combined organic layers were dried andconcentrated to give 4-(3,3-difluoro-propyl)-piperidine-1,2-dicarboxylicacid 1-tert-butyl ester as a clear syrup (163 mg, 88%)

¹H NMR (300 MHz, CDCl₃) δ 5.77 (dddd, J=4.2, 4.2, 56.6, 56.6, 1), 4.34(t, J=6.4, 1), 3.62–3.50 (m, 1), 3.41–3.30 (m, 1), 2.05–1.96 (m, 1),1.92–1.73 (m, 4), 1.70–1.60 (m, 1), 1.52–1.32 (m, 3), 1.43 (s, 9); MS(ESPOS): 330.5 [M+Na]⁺; MS (ESNEG): 306.5 [M−H]⁻.

To a mixture of the HCl salt of compound 2b (R¹=Me, R²=Me) (140 mg, 0.49mmol, 1 equiv) in dry DMF (1.2 mL) at 0° C. was added triethylamine(0.34 mL, 2.43 mmol, 5 equiv), followed by the addition of BSTFA (0.20mL, 0.74 mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for10 minutes, and then was stirred at rt for 50 minutes. To the reactionmixture were added the4-(3,3-Difluoro-propyl)-piperidine-1,2-dicarboxylic acid 1-tert-butylester (153 mg, 0.50 mmol, 1.0 equiv) and HATU (235 mg, 0.62 mmol, 1.26equiv). The reaction mixture was stirred at rt for 3 h. The reactionmixture was evaporated to dryness, taken up in ethyl acetate, washedwith 10% citric acid (1×), water (1×), sat. NaHCO₃ (1×) and brine. Theorganic layer was dried over Na₂SO₄ and evaporated to give a pink syrupwhich was used without purification.

To a solution of the above syrup in DCM (15 mL) with methyl sulfide(0.33 mL) were added trifluoroacetic acid (5 mL) and water (0.33 mL).The reaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (lower isomer,93 mg, 43%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.90 (dddd, J=4.2, 4.2, 56.7, 56.7, 1), 5.24(d, J=6, 1), 4.21 (dd, J=3.5, 9.8, 1), 4.11–4.04 (m, 2), 3.84–3.77 (m,2), 3.51 (dd, J=3.2, 10.3, 1), 3.45–3.37 (m, 1), 3.07–2.98 (m, 1),2.23–2.12 (m, 2), 2.11 (s, 3), 1.98–1.66 (m, 4), 1.52–1.26 (m, 4),0.94–0.88 (m, 6). MS (ESPOS): 441.7 [M+H]⁺.

Example 42 Preparation of 4-(4,4-Difluoro-butyl)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of methyl sulfoxide (0.58 mL, 8.16 mmol, 2.4 equiv) indichloromethane (1.8 mL) at −72° C. was added a 2 M solution of oxalylchloride in dichloromethane (2.04 mL, 4.08 mmol, 1.2 equiv) over aperiod of 1 minute. The mixture was stirred at −72° C. for 25 minutes,followed by the dropwise addition of a solution of the alcohol 14c(R⁹=4-hydroxybutyl), prepared using the procedures in general Method R,(0.71 g, 3.4 mmol, 1 equiv) in dichloromethane (4.8 mL) over a period of2 minutes. The reaction mixture was stirred at −72° C. for 25 minutes,then warmed to −50° C. and stirred for an additional 2 h. Triethylamine(1.89 mL, 13.6 mmol, 4.0 equiv) was added and stirred at −50° C. for 25minutes. The mixture was diluted with ethyl acetate, washed with water(1×), sat. aqueous NaHCO₃ (1×), brine (1×), dried, evaporated andco-evaporated with anhydrous toluene to give aldehyde4-(4-oxo-butyl)-pyridine-2-carboxylic acid methyl ester as an oil (0.66mg, 94%).

¹H NMR (300 MHz, CDCl₃) δ 9.78 (s, 1), 8.62 (d, J=5.1, 1), 7.97 (s, 1),7.29 (d, J=5.1, 1), 3.99 (s, 3), 2.72 (t, J=7.8, 2), 2.50 (t, J=7.2, 2),2.04–1.93 (m, 2); MS (ESPOS): 230.4 [M+Na]⁺.

To a solution of 4-(4-oxo-butyl)-pyridine-2-carboxylic acid methyl ester(0.66 g, 3.19 mmol, 1 equiv) in DCM (12 mL) at −78° C. was added DAST(1.69 mL, 12.75 mmol, 4 equiv). The mixture was warmed to rt and stirredovernight. The mixture was diluted with dichloromethane, washed withsat. aqueous NaHCO₃ (1×), brine (1×), dried, evaporated. The residue waspurified by chromatography to provide4-(4,4-difluorobutyl)-pyridine-2-carboxylic acid methyl ester (0.54 g,74%).

¹H NMR (300 MHz, CDCl₃) δ 8.62 (d, J=5.1, 1), 7.97–7.95 (m, 1),7.29–7.26 (m, 1), 5.81 (dddd, J=3.9, 3.9, 56.6, 56.6, 1), 3.98 (s, 3),2.74 (t, J=7.2, 2), 1.93–1.77 (m, 4). MS (ESPOS): 230.4 [M+H]⁺, 252.4[M+Na]⁺.

To a mixture 4-(4,4-difluorobutyl)-pyridine-2-carboxylic acid methylester (0.54 g, 2.36 mmol, 1 equiv) in MeOH (8 mL) and water (8 mL) wereadded conc. HCl (0.59 mL, 7.07 mmol, 3 equiv) and platinum oxide (0.2g). The mixture was purged and charged with hydrogen (1 atm) and stirredovernight. The platinum oxide was removed by filtration and the filtratewas evaporated to give a residue:

MS (ESPOS): 236.6 [M+H]⁺.

To the residue prepared above were added 2N NaOH (4.72 mL) and t-butylalcohol (2.5 mL). The mixture was stirred at rt for 2 hrs. Thendi-t-butyl dicarbonate (0.77 g, 3.54 mmol) was added. The mixture wasstirred at rt overnight. The solvent was removed under vacuum. Theresidue was diluted with water (10 mL), was washed with ether (20 mL).The aqueous layer was acidified with 2N HCl to pH=2.0, extracted withethyl acetate (2×). The combined organic layers were dried andconcentrated to give 4-(4,4-difluoro-butyl)-piperidine-1, 2-dicarboxylicacid 1-tert-butyl ester (0.67 g, 89%).

¹H NMR (300 MHz, CDCl₃) δ 5.77 (dddd, J=4.3, 4.3, 56.8, 56.8, 1), 4.30(t, J=6.8, 1), 3.58–3.47 (m, 1), 3.41–3.31 (m, 1), 2.05–1.96 (m, 1),1.87–1.68 (m, 4), 1.65–1.56 (m, 1), 1.51–1.30 (m, 5), 1.43 (s, 9); MS(ESPOS): 344.5 [M+Na]⁺.

To a mixture of the HCl salt of compound 2b (R¹=Me, R²=Me) (153 mg, 0.53mmol, 1 equiv) in dry DMF (1.3 mL) at 0° C. was added triethylamine(0.37 mL, 2.66 mmol, 5 equiv), followed by the addition of BSTFA (0.21mL, 0.80 mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for10 minutes, and then was stirred at rt for 50 minutes. To the reactionmixture were added the4-(4,4-difluoro-butyl)-piperidine-1,2-dicarboxylic acid 1-tert-butylester (196 mg, 0.61 mmol, 1.15 equiv) and HATU (293 mg, 0.77 mmol, 1.45equiv). The reaction mixture was stirred at rt for 3 h. The reactionmixture was evaporated to dryness, taken up in ethyl acetate, washedwith 10% citric acid (1×), water (1×), sat. NaHCO₃ (1×) and brine. Theorganic layer was dried over Na₂SO₄ and evaporated to give the crudeproduct as a syrup. The residue was dissolved in methanol (20 mL), thendried and washed Dowex™ resin (100 mg) was added. The mixture wasstirred at rt for 30 minutes, and filtered. The filtrate wasconcentrated to give a clear syrup, which was purified by chromatographyto give a clear syrup (0.25 g, 85%).

MS (ESPOS): 555.8 [M+H]⁺.

To a solution of the above syrup in DCM (15 mL) with methyl sulfide(0.33 mL) were added trifluoroacetic acid (5 mL) and water (0.33 mL).The reaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (lower isomer,70 mg, 34%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.88 (dddd, J=4.4, 4.4, 57, 57, 1), 5.24 (d,J=5.4, 1), 4.20 (dd, J=3.2, 10.1, 1), 4.12–4.03 (m, 2), 3.90–3.80 (m,2), 3.52 (dd, J=3.5, 10.3, 1), 3.46–3.39 (m, 1), 3.09–2.98 (m, 1),2.25–2.12 (m, 2), 2.11 (s, 3), 1.98–1.67 (m, 4), 1.56–1.30 (m, 6),0.95–0.87 (m, 6); MS (ESPOS): 455.7 [M+H]⁺.

Example 43 Preparation of4-(5,5-Difluoro-pentyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

Method R is used to prepare compound 14c (R^(9′)=5-hydroxypentyl) (To adry flask was added compound 14a (2 g, 7.60 mmol, 1 equiv),triphenylphosphine (159.4 mg, 0.61 mmol, 0.08 equiv), copper (I) iodide(115.8 mg, 0.61 mmol, 0.08 equiv), palladium acetate (68.2 mg, 0.30mmol, 0.04 equiv) and triethylamine (28 mL). The mixture was deaeratedwith nitrogen, followed by addition of 4-pentyn-1-ol (1.28 g, 15.21mmol, 2 equiv). The mixture was stirred at rt overnight. The solvent wasremoved under vacuum to give a dark residue. The residue was purified bychromatography to give 14b (R^(9′)=5-hydroxypent-1-yn-yl).

To a solution of 14b (R^(9′)=5-hydroxypent-1-yn-yl) in methanol (60 mL)was added 10% palladium on carbon (0.62 g). The mixture was purged andcharged with hydrogen (1 atm) and stirred at rt overnight. The palladiumwas removed by filtration and the filtrate was concentrated to give ayellow oil 14c (R⁹=5-hydroxypentyl) (1.34 g, 79%).

¹H NMR (300 MHz, CDCl₃) δ 8.62 (d, J=4.8, 1), 7.97 (s, 1), 7.31 (dd,J=1.6, 5, 1), 3.99 (s, 3), 3.63 (t, J=6.5, 2), 2.70 (t, J=7.7, 2),1.74–1.53 (m, 4), 1.46–1.34 (m, 2).

To a solution of methyl sulfoxide (0.46 mL, 6.42 mmol, 2.6 equiv) indichloromethane (1.4 mL) at −72° C. was added a 2 M solution of oxalylchloride in dichloromethane (1.61 mL, 3.21 mmol, 1.3 equiv) over aperiod of 1 minute. The mixture was stirred at −72° C. for 25 minutes,followed by the dropwise addition of a solution of pyridine 14c(R⁹=5-hydroxypentyl) (0.55 g, 2.47 mmol, 1 equiv) in dichloromethane(3.8 mL) over a period of 2 minutes. The reaction mixture was stirred at−72° C. for 25 minutes, then warmed to −50° C. and stirred for anadditional 2 h. Triethylamine (1.48 mL, 10.7 mmol, 4.33 equiv) was addedand stirred at −50° C. for 25 minutes. The mixture was diluted withethyl acetate, washed with water (2×), sat. aqueous NaHCO₃ (1×), brine(1×), dried, evaporated and co-evaporated with anhydrous toluene to4-(5-oxo-pentyl)-pyridine-2-carboxylic acid methyl ester (0.48 mg, 88%).

¹H NMR (300 MHz, CDCl₃) δ 9.75 (t, J=1.4, 1), 8.61 (d, J=5.1, 1),7.97–7.95 (m, 1), 7.28 (dd, J=1.7, 5, 1), 3.99 (s, 3), 2.73–2.67 (m, 2),2.50–2.44 (m, 2), 1.71–1.63 (m, 4).

To a solution of the to 4-(5-oxo-pentyl)-pyridine-2-carboxylic acidmethyl ester oil (0.48 g, 2.19 mmol, 1 equiv) in DCM (8 mL) at −78° C.was added DAST (1.41 g, 8.74 mmol, 4 equiv). The mixture was warmed tort and stirred overnight. The mixture was diluted with dichloromethane,washed with sat. aqueous NaHCO₃ (1×), dried, and evaporated. The residuewas purified by chromatography to4-(5,5-Difluoro-pentyl)-pyridine-2-carboxylic acid methyl ester (278 mg,52%)

¹H NMR (300 MHz, CDCl₃) δ 8.61 (dd, J=0.6, 4.8, 1), 7.97–7.95 (m, 1),7.28 (dd, J=1.5, 4.8, 1), 5.78 (dddd, J=4.3, 4.3, 57, 57, 1), 3.99 (s,3), 2.70 (t, J=7.7, 2), 1.94–1.66 (m, 4), 1.55–1.43 (m, 2).

MS (ESPOS): 244.2 [M+H]⁺

To a mixture of 4-(5,5-difluoro-pentyl)-pyridine-2-carboxylic acidmethyl ester (278 mg, 1.14 mmol) in MeOH (5 mL) and water (5 mL) wereadded conc. HCl (0.286 mL, 3.43 mmol, 3 equiv) and platinum oxide (140mg). The mixture was purged and charged with hydrogen (1 atm) andstirred overnight. The platinum oxide was removed by filtration and thefiltrate was evaporated to4-(5,5-difluoro-pentyl)-piperidine-2-carboxylic acid 2-methyl ester.

MS (ESPOS): 250.2 [M+H]⁺.

To the above residue 4-(5,5-difluoro-pentyl)-piperidine-2-carboxylicacid-2-methyl ester were added 2N NaOH (2.3 mL) and t-butyl alcohol (1.2mL). The mixture was stirred at rt for 2 hrs. Then di-t-butyldicarbonate (0.37 g, 1.72 mmol) was added. The mixture was stirred at rtovernight. The solvent was removed under vacuum. The residue was dilutedwith water, was washed with ether. The aqueous layer was acidified with2N HCl to pH=2.0, extracted with ethyl acetate (2×). The combinedorganic layers were dried and concentrated to4-(5,5-Difluoro-pentyl)-piperidine-1,2-dicarboxylic acid 1-tert-butylester (310 mg, 81%).

¹H NMR (300 MHz, CD₃OD) δ 5.84 (dddd, J=4.5, 4.5, 57, 57,1), 4.31 (t,J=6.3, 1), 3.65–3.56 (m, 1), 3.35–3.25 (m, 1), 2.03–1.63 (m, 5),1.48–1.30 (m, 8), 1.43 (s, 9).

To a mixture of the HCl salt of compound 2b (R¹=Me, R²=Me) (223.7 mg,0.78 mmol, 1 equiv) in dry DMF (1.9 mL) at 0° C. was added triethylamine(0.54 mL, 3.89 mmol, 5 equiv), followed by the addition of BSTFA (0.31mL, 1.17 mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for10 minutes, and then was stirred at rt for 50 minutes. To the reactionmixture were added 4-(5,5-difluoro-pentyl)-piperidine-1,2-dicarboxylicacid 1-tert-butyl ester (272 mg, 0.81 mmol, 1.05 equiv) and HATU (391mg, 1.03 mmol, 1.32 equiv). The reaction mixture was stirred at rt for 3h. The reaction mixture was evaporated to dryness, taken up in ethylacetate, washed with 10% citric acid (1×), water (1×), sat. NaHCO₃ (1×)and brine. The organic layer was dried over Na₂SO₄ and evaporated togive a residue. The residue was dissolved in methanol (30 mL), then dryand washed Dowex™ resin (150 mg) was added. The mixture was stirred atrt for 1 h and filtered. The filtrate was concentrated to give a clearsyrup, which was purified by chromatography to give a clear syrup (0.26g, 72%).

To a solution of the above syrup in DCM (15 mL) with methyl sulfide(0.33 mL) were added trifluoroacetic acid (5 mL) and water (0.33 mL).The reaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (lower isomer,40 mg, 15%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.86 (dddd, J=4.5, 4.5, 57, 57, 1), 5.24 (d,J=5.7, 1), 4.21 (dd, J=3.3, 9.9, 1), 4.11–4.04 (m, 2), 3.86–3.78 (m, 2),3.51 (dd, J=3.5, 10.4, 1), 3.47–3.38 (m, 1), 3.07–2.97 (m, 1), 2.23–2.12(m, 2), 2.11 (s, 3), 1.98–1.64 (m, 4), 1.50–1.27 (m, 8), 0.94–0.87 (m,6); MS (ESPOS): 469.4 [M+H]⁺.

Example 44 Preparation of 4-(5-Fluoro-pentyl)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of compound 14c (R⁹=5-hydroxypentyl) (0.66 g, 2.96 mmol, 1equiv), prepared as described in general Method R and in the synthesisof Example 47, in DCM (11 mL) at −78° C. was added DAST (1.91 g, 11.85mmol, 4 equiv). The mixture was warmed to rt and stirred overnight. Themixture was diluted with dichloromethane, washed with sat. aqueousNaHCO₃ (1×), dried, and evaporated. The residue was purified bychromatography to give 4-(5-fluoro-pentyl)-pyridine-2-carboxylic acidmethyl ester (254 mg, 38%)

¹H NMR (300 MHz, CDCl₃) δ 8.62 (d, J=4.8, 1), 7.97 (d, J=1.2, 1), 7.30(dd, J=1.7, 5, 1), 4.50 (t, J=5.9, 1), 4.34 (t, J=6, 1), 3.99 (s, 3),2.70 (t, J=7.7, 2), 1.80–1.62 (m, 4), 1.50–1.41 (m, 2).

To a mixture of 4-(5-fluoro-pentyl)-pyridine-2-carboxylic acid methylester (254 mg, 1.13 mmol) in MeOH (5 mL) and water (5 mL) were addedconc. HCl (0.28 mL, 3.39 mmol, 3 equiv) and platinum oxide (130 mg). Themixture was purged and charged with hydrogen (1 atm) and stirredovernight. The platinum oxide was removed by filtration and the filtratewas evaporated to give 4-(5-fluoro-pentyl)-piperidine-2-carboxylic acid2-methyl ester.

MS (ESPOS): 232.4 [M+H]⁺.

To 4-(5-fluoro-pentyl)-piperidine-2-carboxylic acid 2-methyl ester wasadded 2N NaOH (2.43 mL) and t-butyl alcohol (1.3 mL). The mixture wasstirred at rt for 2 hrs. Then di-t-butyl dicarbonate (0.40 g, 1.82 mmol)was added. The mixture was stirred at rt overnight. The solvent wasremoved under vacuum. The residue was diluted with water, was washedwith ether. The aqueous layer was acidified with 2N HCl to pH=2.0,extracted with ethyl acetate (2×). The combined organic layers weredried and concentrated to give4-(5-fluoro-pentyl)-piperidine-1,2-dicarboxylic acid 1-tert-butyl esteras a syrup (254 mg, 71%).

¹H NMR (300 MHz, CDCl₃) δ 4.52–4.06 (m, 3), 3.55–3.30 (m, 2), 2.03–1.94(m, 1), 1.81–1.54 (m, 4), 1.45–1.20 (m, 8), 1.43 (s, 9). MS (ESPOS):218.3 [M+Na−Boc]⁺

To a mixture of the HCl salt of compound 2b (R¹=Me, R²=Me) (213.8 mg,0.74 mmol, 1 equiv) in dry DMF (1.8 mL) at 0° C. was added triethylamine(0.52 mL, 3.72 mmol, 5 equiv), followed by the addition of BSTFA (0.30mL, 1.12 mmol, 1.5 equiv). The reaction mixture was stirred at 0° C. for10 minutes, and then was stirred at rt for 50 minutes. To the reactionmixture were added the 4-(5-fluoro-pentyl)-piperidine-1,2-dicarboxylicacid 1-tert-butyl ester as a syrup (244 mg, 0.77 mmol, 1.04 equiv) andHATU (370 mg, 0.97 mmol, 1.31 equiv). The reaction mixture was stirredat rt for 3 h. The reaction mixture was evaporated to dryness, taken upin ethyl acetate, washed with 10% citric acid (1×), water (1×), sat.NaHCO₃ (1×) and brine. The organic layer was dried over Na₂SO₄ andevaporated to give a residue. The residue was dissolved in methanol (30mL), then dry and washed Dowex™ resin (140 mg) was added. The mixturewas stirred at rt for 1 h and filtered. The filtrate was concentrated togive a clear syrup, which was purified by chromatography to give a clearsyrup (212 mg, 52%).

To a solution of the above syrup in DCM (15 mL) with methyl sulfide(0.33 mL) were added trifluoroacetic acid (5 mL) and water (0.33 mL).The reaction mixture was stirred at rt for 1 h. The solvent was removedunder vacuum and co-evaporated with toluene twice. The residue waspurified by chromatography to provide the title compound (lower isomer,40 mg, 17%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.49 (t, J=5.9, 1), 4.33(t, J=6, 1), 4.20 (dd, J=3.5, 10.1, 1), 4.11–4.04 (m, 2), 3.83–3.77 (m,2), 3.51 (dd, J=3.3, 10.2, 1), 3.44–3.36 (m, 1), 3.06–2.94 (m, 1),2.23–2.13 (m, 2), 2.11 (s, 3), 1.98–1.88 (m, 1), 1.77–1.59 (m, 3),1.45–1.27 (m, 8), 0.94–0.87 (m, 6). 04261 MS (ESPOS): 451.4 [M+H]⁺

Example 45 Preparation of 4-(4-Fluoro-butyl)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a solution of compound 14c (R⁹=4-hydroxybutyl) (0.76 g, 3.62 mmol, 1equiv), prepared as described in general Method R and in the synthesisof Example 47, in DCM (14 mL) at −78° C. was added DAST (1.9 mL, 14.47mmol, 4 equiv). The mixture was warmed to rt and stirred overnight. Themixture was diluted with dichloromethane, washed with sat. aqueousNaHCO₃ (1×), brine (1×), dried, evaporated. The residue was purified bychromatography to provide 4-(4-Fluoro-butyl)-pyridine-2-carboxylic acidmethyl ester as a yellow oil (0.24 g, 31%).

¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J=4.8, 1), 7.92 (d, J=1.2, 1),7.27–7.23 (m, 1), 4.49 (t, J=5.6, 1), 4.33 (t, J=5.6, 1), 3.94 (s, 3),2.69 (t, J=7.5, 2), 1.79–1.59 (m, 4).

To a mixture of 4-(4-fluoro-butyl)-pyridine-2-carboxylic acid methylester (0.24 g) in THF (3 mL) and water (1 mL) was added lithiumhydroxide monohydrate (71.3 mg, 1.7 mmol, 1.5 equiv). The mixture wasstirred at rt overnight and diluted with methanol (20 mL). Then H⁺ resinwas added and the mixture was shaken for 10 minutes. The resin waswashed with methanol (1×), 1:1 acetonitrile/water (1×), and acetonitrile(1×). The product was eluted with 5% TEA in methanol (4×) andacetonitrile (1×). The combined organic solvents were evaporated andco-evaporated with toluene to provide4-(4-Fluoro-butyl)-pyridine-2-carboxylic acid (0.22 g, 65%).

¹H NMR (300 MHz, CD₃OD) δ 8.46 (d, J=4.8, 1), 7.95 (s, 1), 7.39–7.35 (m,1), 4.52 (t, J=5.6, 1), 4.36 (t, J=5.9, 1), 3.22 (q, J=7.3, 2.5H, TEA),2.77 (t, J=7.5, 2), 1.84–1.62 (m, 4), 1.28 (t, J=7.2, 3.8H, TEA).

To a solution of 4-(4-fluoro-butyl)-pyridine-2-carboxylic acid (0.22 g,0.73 mmol, 1 equiv) in dry acetonitrile (4 mL) at 0° C. was addedtriethylamine (74 mg, 0.73 mmol, 1 equiv), followed by the addition ofisobutyl chloroformate (100 mg, 0.73 mmol, 1 equiv). The reactionmixture was stirred at 0° C. for 15 minutes, and then was stirred at 40C for 2 h. To the reaction mixture was added a solution of the HCl saltof compound 2b (R¹=Me, R²=Me) (263 mg, 0.91 mmol, 1.25 equiv) andtriethylamine (93 mg, 0.91 mmol, 1.25 equiv) in a 1:1 acetone/water (4mL). The reaction mixture was stirred at 4° C. overnight. The reactionmixture was evaporated to dryness, taken up in DCM, washed with sat.NaHCO₃ (1×). The organic layer was dried over Na₂SO₄ and evaporated. Theresidue was purified by chromatography to give a clear solid (110 mg,35%).

To a solution of the above solid (110 mg, 0.25 mmol, 1 equiv) in MeOH (6mL) and water (4 mL) were added conc. HCl (20.2 uL, 0.24 mmol, 0.95equiv) and platinum oxide (220 mg). The mixture was purged and chargedwith hydrogen (65 psi) and shaken overnight. The platinum oxide wasremoved by filtration and the filtrate was evaporated to give a residue,which was purified by chromatography to provide the title compound(lower isomer, 33 mg, 30%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.4, 1), 4.51 (t, J=6, 1), 4.35 (t,J=5.9, 1), 4.21 (dd, J=3.3, 10.2, 1), 4.10–4.04 (m, 2), 3.93–3.80 (m,2), 3.52 (dd, J=3.3, 10.2, 1), 3.46–3.38 (m, 1), 3.11–2.98 (m, 1),2.26–2.13 (m, 2), 2.11 (s, 3), 2.00–1.92 (m, 1), 1.80–1.60 (m, 3),1.54–1.27 (m, 6), 0.95–0.87 (m, 6).

MS (ESPOS): 437.4 [M+H]⁺

Example 46 Preparation of4-(3-Ethyl-3-hydroxy-pentyl)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a dry flask was added compound 13b (R¹=Me, R²=Me, and R³=H) madeusing general Method Q (130 mg, 0.27 mmol, 1 equiv), triphenylphosphine(45.3 mg, 0.17 mmol, 0.64 equiv), copper (I) iodide (32.9 mg, 0.17 mmol,0.64 equiv), palladium acetate (19.4 mg, 0.086 mmol, 0.32 equiv) andtriethylamine (1.5 mL). The mixture was deaerated with nitrogen,followed by addition of 3-ethyl-1-pentyn-3-ol (174 uL, 1.35 mmol, 5equiv). The mixture was stirred at 50° C. overnight. The solvent wasremoved under vacuum to give a dark residue. The residue was purified bychromatography to give 13c (R¹=Me, R²=Me, R³=H,R⁹=3-Ethyl-3-hydroxy-pent-1-ynyl).

MS (ESPOS):. 467.7 [M+H]⁺; MS (ESNEG): 465.5 [M−H]⁻.

To a mixture of the above syrup in MeOH (12 mL) and water (8 mL) wereadded platinum oxide (300 mg) and conc. HCl (26 uL). The mixture waspurged and charged with hydrogen (65 psi) and shaken overnight. Theplatinum oxide was removed by filtration and the filtrate wasevaporated. The residue was purified by chromatography to give the titlecompound as a white solid (19 mg, 15%).

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7, 1), 4.17 (dd, J=3.1, 10.0, 1),4.10–4.02 (m, 2), 3.80 (d, J=3, 1), 3.53–3.48 (m, 1), 3.42–3.35 (m, 1),3.23–3.15 (m, 1), 2.75–2.64 (m, 1), 2.22–2.11 (m, 1), 2.10 (s, 3),2.04–1.97 (m, 1), 1.80–1.72 (m, 1), 1.50–1.40 (m, 6), 1.31–1.06 (m, 5),0.94–0.80 (m, 12); MS (ESPOS): 477.8 [M+H]⁺; MS (ESNEG): 475.6 [M−H]⁻.

Example 47 Preparation of 4-Butoxy-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To trimethylsilyl cyanide (5.2 g, 52 mmol), 4-benzyloxy-pyridine 1-oxide(8.8 g, 44 mmol) in DCM (20 mL) was added, followed by dimethylcarbamoylchloride (5.6 g, 52 mmol) in DCM (10 mL), dropwise, stirred at roomtemperature overnight. Sodium bicarbonate (100 mL, 10%) was added,stirred for 10 minutes and extracted twice with DCM (50 mL). Thecombined organic layer was dried over magnesium sulfate, solvent wasremoved to obtain the product, compound 10a (R⁹=Benzyloxy) (10.5 g,100%).

¹H NMR (300 MHz, CDCl₃) δ 8.34 (d, J=5.7, 1), 7.24 (m, 5), 7.11 (t,J=2.4, 1), 6.90 (dd, J=5.7, 2.4, 1), MS (ES+): 211(M+1).

Compound 10a (R⁹=Benzyloxy) (5 g, 23 mmol) was dissolved in HCl (6N, 70mL) and refluxed overnight. The crude product4-hydroxypyridine-2-carboxylic acid, compound 10b (R⁹=hydroxy) obtainedon removal of HCl was crystallized from acetonitrile (2.6 g, 80%).

¹H NMR (300 MHz, CD₃OD) δ 8.55 (d, J=6.6, 1), 7.78 (d, J=3.0, 1), 6.90(dd, J=2.7, 6.9, 1), MS (ES⁻): 138 (M−1).

The synthesis of title compound was completed using the syntheticsequence found in general Method S starting from4-hydroxypyridine-2-carboxylic acid, as prepared above.

¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.4, 1), 4.22 (dd, J=10.2; 3.3, 1),4.08 (m, 2), 3.81 (d, J=3.0, 1), 3.70 (m, 1), 3.54 (m, 4), 3.43 (m, 2),2.90 (m, 1), 2.41 (m, 1), 2.19 (m, 1), 2.10 (s, 3) 1.45 (m, 6), 0.92 (m,9); MS (ES+): 435 (M+1).

Example 48 Preparation of 4-Pentyloxy-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the synthetic sequence found ingeneral Method S starting from 4-hydroxypyridine-2-carboxylic acid 10b(R⁹=hydroxy) substituting n-pentyl bromide as the alkylating agent.

Compound 15a (R¹⁰=pentyl): ¹H NMR (300 MHz, CD₃OD) δ 8.38 (d, J=5.1, 1),7.64 (s, 1), 7.10 (d, J=3.3, 1), 4.18 (t, J=6.6, 2), 1.85 (m, 2), 1.49(m, 4), 0.96 (t, J=7.2, 3). MS (ES⁻): 208(M−1).

Compound 15b (R¹=Me, R²=Me, R¹⁰=butyl): ¹H NMR (300 MHz, CD₃OD) δ 8.41(d, J=5.7, 1), 7.61 (d, J=2.4, 1), 7.07 (dd, J=2.4, 5.4, 1), 5.27 (d,J=5.4, 1), 4.05–4.31 (m, 5), 3.85 (d, J=3.0, 1), 3.57 (dd, J=3.3, 7.2,1), 2.11 (m, 4), 1.81 (m, 2), 1.49 (m, 4), 1.00 (m, 9). MS (ES+): 443(M+1).

Title compound (20 mg, 10%): ¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7,1), 4.22 (dd, J=9.9; 3.3, 1), 4.10 (m, 2), 3.76 (m, 3), 3.51 (m, 3),3.39 (m, 1), 3.02 (m, 2), 2.43 (m, 1), 2.15 (m, 1), 2.10 (s, 3) 1.95 (m,2), 1.69 (m, 2), 1.53 (m, 2), 1.34 (m, 2), 0.93 (m, 9); MS (ES+): 449(M+1).

Example 49 Preparation of 4-(4-Fluoro-butoxy)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the synthetic sequence found ingeneral Method S starting from 4-hydroxypyridine-2-carboxylic acid 10b(R⁹=hydroxy) substituting 1-bromo-4 -fluoro-butane as the alkylatingagent.

¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.7, 1), 4.53 (t, J=5.7, 1), 4.37(t, J=5.7, 1), 4.21 (dd, J=3.3, 6.6, 1), 4.07 (m, 2), 3.80 (d, J=3.3,2), 3.60 (m, 5), 2.88 (m, 1), 2.38 (m, 1), 2.18 (m, 1), 2.10 (s, 3)1.33–1.83 (m, 8), 0.92 (m, 6); MS (ES⁺): 453 (M+1).

Example 50 Preparation of 4-Butyl-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-allyl]-amide

To a solution of Boc 7-Methylene MTL (P=Boc, R¹=Me, R^(2′)=CH₂) preparedfrom compound 2a (P=Boc, R¹=Me) by general Method D (391 mg, 1.1 mmol)in a solution of dichloroethane (10 mL) and dimethylsulfide (0.4 mL, 2.5mmol) was added, TFA (5 mL) containing water (0.4 mL) and the reactionmixture stirred at rt for 45 min. The solvent was removed and theresidue evaporated twice from DCE to obtain the crude product. Theproduct was obtained as an HCl salt by precipitation from ethyl acetate(4 mL) at 0° C. by addition of 2M HCl in ether, and dried under vacuum(351 mg g, 86%). The white solid product was used in the next reactionwithout additional purification.

MS (ESPOS): 350 (M+H).

Triethylamine (0.68 mL, 4.9 mmol, 4.0 equiv), followed by BSTFA (0.58mL, 2.20 mmol, 1.8 equiv), were added to a stirred suspension ofcompound prepared above (351 mg, 1.22 mmol, 1 equiv) in anhydrous DMF (5mL) at 0° C. and under nitrogen. The resulting mixture was stirred at 0°C. for 10 min, and then at rt for 50 min. The resulting solution wascooled to 0° C. and a solution of compound 7d (R⁹=n-butyl) prepared byMethod K (400 mg, 1.47 mmol, 1.2 equiv) in anhydrous DMF (5 mL) wasadded, followed by solid HATU (741 mg, 1.95 mmol, 1.6 equiv). Thereaction mixture was allowed to warm to rt and after 2 h the reactionsolution was evaporated to dryness under vacuum. The residual oilobtained was diluted with EtOAc (200 mL), washed sequentially with 10%citric acid, 1:1. saturated aqueous NaHCO₃, water, and brine dried overNa₂SO₄, and evaporated to dryness.

To a solution of 50 mg crude coupling product in 1,2-dichloroethane (6mL), was added dimethylsulfide (200 μL), followed by TFA (11.5 mL), andwater (768 μL). The resulting mixture was stirred at rt for 1 h,evaporated to a minimal volume, diluted with DCE (3×10 mL), andevaporated to dryness. The residue was purified by column chromatography8% to 12% 0.25M methanolic ammonia in dichloromethane to provide thetitle compound (10.0 mg, 25%).

¹H NMR (300 MHz, CD₃OD) δ 5.22 (d, J=5.8, 1), 5.00 (s, 1), 4.95 (s, 1),4.58 (d, J=8.8, 1) 4.19 (d, J=8.8, 1), 4.09 (dd, J=5.8, 10.1, 1)3.85–3.77 (m, 2), 3.57–3.52 (m, 1), 3.26–3.29 (m, 1), 2.59–2.53 (m, 1),2.10–1.98 (m, 4), 1.80 (s, 3), 1.36–1.51–1.11 (m, 7), 0.91 (t, J=6.9,3); MS (ESPOS): 403.6 [M+H]⁺.

Example 51 Preparation of 1,4-Diethyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To the product of Example 1 (30 mg, 0.07 mmol) in DMF (1 mL), DIEA (43mg, 0.35 mmol) was added at room temperature and stirred overnight. Thenremoved the solvent and the resulting product was purified by columnchromatography using 20% MeOH in DCM to obtain the title compound (20mg, 66%) as a white powder.

¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=6.0, 1), 4.26 (dd, J=3.6, 9.6, 1),3.79 (d, J=3.0, 1), 3.55 (dd, J=3.3, 10.2, 1) 2.85 (m, 2), 2.13 (m, 4),1.37 (m, 12), 0.94 (m, 9); MS (ES+): 420 (M+1).

Example 52 Preparation of 4-(3-Fluoro-propoxy)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the synthetic sequence found ingeneral Method S starting from 4-hydroxypyridine-2-carboxylic acidsubstituting 1-bromo-3-fluoro-propane as the alkylating agent.

Compound 15a (R¹⁰=3-fluoropropyl): ¹H NMR (300 MHz, CD₃OD) δ 8.41 (d,J=5.1, 1), 7.65 (d, J=2.1, 1), 7.14 (dd, J=2.1, 5.7, 1), 4.59 (m, 2),4.24 (t, J=6.0, 2), 1.91 (m, 2). MS (ES−): 212(M−1).

Compound 15b (R¹=Me, R²=Me, R¹⁰=3-fluoropropyl): ¹H NMR (300 MHz, CD₃OD)δ 8.44 (d, J=5.7, 1), 7.65 (d, J=2.4, 1), 7.12 (dd, J=2.4, 5.7, 1), 5.48(d, J=5.7, 1), 4.87 (m, 2), 4.30 (m, 2), 4.12 (dd, J=3.0, 10.2, 1), 3.85(d, J=3.3, 1), 3.56 (dd, J=9.9, 3.3, 1), 2.26 (m, 1), 2.11 (s, 3), 1.37(m, 4), 1.00 (t, J=5.1, 6). MS (ES+): 443 (M+1).

Title compound (60 mg, 31%): ¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.7,1), 4.50 (m, 2), 4.21 (dd, J=3.3, 9.9, 1), 4.06 (m, 2), 3.80 (d, J=2.7,1), 3.66 (m, 3), 3.59 (m, 1), 3.33 (m, 1), 2.87 (m, 1), 2.41 (m, 1),2.18 (m, 1), 2.10 (s, 3) 1.91 (m, 4), 1.51 (m, 2), 0.92 (m, 6); MS(ES+): 439 (M+1).

Example 53 Preparation of4-(3,3,3-Trifluoro-propoxy)-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the synthetic sequence found ingeneral Method S starting from 4-hydroxypyridine-2-carboxylic acidsubstituting 2-trifluoroethyl bromide as the alkylating agent.

Compound 15a (R¹⁰=2-trifluoroethyl): ¹H NMR (300 MHz, CD₃OD) δ 8.67 (m,1), 7.92 (s, 1), 7.43 (m, 1), 4.65 (m, 2), 3.01 (m, 2). MS (ES−):234(M−1).

Compound 15b (R¹=Me, R²=Me, R¹⁰=2-trifluoroethyl): ¹H NMR (300 MHz,CD₃OD) δ 8.46 (d, J=6.0, 1), 7.65 (d, J=2.7, 1), 7.13 (dd, J=2.7, 6.0,1), 5.27 (d, J=5.7, 1), 4.39 (t, J=6.0,2), 4.30 (m, 2), 4.11 (m, 1),3.85 (d, J=3.0, 1), 3.57 (dd, J=3.0, 10.2, 1), 2.88 (m, 2), 2.25 (m, 1),2.11 (s, 3), 1.00 (t, J=6.9, 6). MS (METHOD ES+): 469 (M+1).

Title compound (10 mg, 10%): ¹H NMR (300 MHz, CD₃OD) δ 5.24 (d, J=5.7,1), 4.18 (dd, J=3.0, 9.9, 1), 4.15 (m, 2), 3.80 (d, J=3.6, 1), 3.74 (m,2), 3.52 (dd, J=3.3, 10.2, 2), 3.38 (m, 2), 3.18 (m, 1), 2.66 (m, 1),2.66 (m, 1), 2.44 (m, 2), 2.22 (m, 1), 2.10 (s, 3) 1.34 (m, 2), 0.91 (d,J=7.2, 6); MS (ES+): 475 (M+1).

Example 54 Preparation of 4-Isobutyl-piperidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

4-Isobutyl-2-cyanopyridine is prepared as follows. Toisobutyltriphenylphosphonium iodide (Aldrich) (8 g, 18.5 mmol) in THF(20 mL) at 0° C., potassium-tert-butoxide (1.8 g, 16 mmol) was added,stirred at room temperature for 1 hr. pyridine-4-carboxlaldehyde (1 g,9.3 mmol) was added, stirred at room temperature for overnight. Reactionmixture was then poured to water (100 mL) and extracted with EtOAc (100mL). The product obtained on removal of solvent was purified by columnchromatography using 40% EtOAc in hexane as eluent (1.05 g, 84%). To theresulting product (4.2 g, 31.5 mmol), 10% Pd/C (0.4 g) was added andhydrogenated at 1 atm pressure overnight. Removal of solvent andpurification on column chromatography using 30% EtOAc in hexanesresulted in 4-isobutylpyridine (3.8 g, 90%).

The intermediate, 4-isobutylpyridine-2-carboxylic acid, compound 10b,(R⁹=isobutyl) was made by employing general Method P. To4-isobutylpyridine (2 g, 14.8 mmol) in acetic acid (20 mL), hydrogenperoxide (3.35 g, 30%, 30 mmol) was added and refluxed overnight. Afterremoving the solvent, the residue was dissolved in DCM dried overmagnesium sulfate and taken as such for the next step. To the compoundin DCM (10 mL) trimethylsilyl cyanide (1.73 g, 17.4 mmol) anddimethylcarbonyl chloride (1.89 g, 17.4 mmol) was added and stirred atroom temperature for 24 hours. Aqueous potassium bicarbonate (100 mL,10%) was added and extracted twice with DCM (50 mL each). The crudeproduct obtained on removal of solvent was taken in HCl (6N, 100 mL) andrefluxed for 24 hours. Removal of acid and purification of crude productby column chromatography using 30% MeOH in DCM resulted in acid 10b(R⁹=isobutyl) (1.5 g, 100%).

¹H NMR (300 MHz, CD₃OD) δ 8.78 (d, J=5.7, 1), 8.44 (s, 1), 8.13, (d,J=5.7, 1), 2.92 (d, J=7.5, 1), 2.15 (m, 1), 0.98 (d, J=6.6, 6). MS.(ES−):. 178(M−1).

To the amine 2b (R¹=Me, R²=Me) (200 mg, 0.79 mmol) in DMF (2 ml), TEA(161 mg, 1.6 mmol), BSTFA (614 mg, 2.4 mmol) was added at 0° C. andstirred at room temperature for 1.5 hr. Acid 10b (R⁹=isobutyl) (214 mg,1.2 mmol) and HATU (368 mg, 1.2 mmol) was added and let stirred at roomtemperature for 4 hours. DMF was removed and the residue was extractedwith EtOAc (50 mL), washed with sodium bicarbonate (10%, 50 mL), brine(50 mL) and dried over magnesium sulfate. The product obtained onremoval of solvent was dissolved in methanol (10 mL) and treated withresin (300 mg) for 3 hr. After filtering the resin, methanol was removedto obtain the crude product. It was then purified on silica gel columnchromatography using 3% MeOH in DCM to obtain compound 11b (R¹=Me,R²=Me, R³=H, R⁹=isobutyl) (200 mg, 60%).

¹H NMR (300 MHz, CD₃OD) δ 8.41. (d, J=4.8, 1), 8.28 (d, J=9.6,1), 7.95(s, 1), 5.35 (d, J=5.4, 1), 4.25 (m, 2), 3.99 (d, J=10.8, 1), 3.78 (d,J=3.6, 1), 3.55 (dd, J=3.6, 10.8, 1), 2.52 (m, 3), 2.15 (s, 3), 1.93 (m,1), 1.02 (m, 12). MS (ES+): 413 (M+1).

To compound 11b (R¹=Me, R²=Me, R³=H, R⁹=isobutyl) (200 mg, 0.48 mmol) inwater (10 mL), AcOH (2 mL) and MeOH (2 mL), PtO₂ (200 mg), was added,hydrogenated at 55 psi for 16 hours. After filtering the catalyst, thesolvent was removed to obtain the crude material which on purificationover silica gel column using 20% MeOH in DCM as eluent. The lower R_(f)fraction provided the title compound (70 mg, 34%).

¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.7, 1), 4.20 (dd, J=9.9; 3.3, 1),4.07 (m, 2), 3.80 (d, J=3.0, 1), 3.60 (m, 2), 3.34 (m, 2), 2.84 (m, 1),2.17 (m, 1), 2.10 (s, 3) 2.01 (m, 1), 1.77 (m, 3), 1.40 (m, 4), 0.91 (m,12); MS (ES+): 419 (M+1).

Example 55 Preparation of 4-Propyl-piperidine-2-carboxylic acid[2,2-difluoro-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-nyran-2-yl)-propyl]-amide

To acid 10b (R⁹=propyl) prepared by Method P (53 mg, 0.32 mmol) in DMF(3 mL), lincosamide intermediate, compound 5b (R¹=Me), prepared byMethod 1 (63. mg, 0.16 mmol) and HATU (121. mg, 0.32 mmol),triethylamine (70 mg, 0.48 mmol) was added at 0° C. and stirred at roomtemperature 16 hours. DMF was removed and the residue was taken in ethylacetate and washed with saturated bicarbonate (30 mL). The productobtained on removal of solvent was purified on silica gel column using30% ethyl acetate in hexanes (40 mg, 45%). To the pure product inmethanol (5 mL), water (5 mL), acetic acid (5 mL) and platinum dioxide(50 mg, mmol) was added and hydrogenated at 60 psi for 16 hours. Afterfiltering the catalyst, the solvent was removed to obtain the crudeproduct which was taken in methanol (3 mL). Potassium carbonate (125 mg,0.83 mmol) in water (1 mL) was added to it and stirred 16 hours.Solvents were then removed and the crude product purified on columnchromatography using 20% methanol in dichloromethane. The lower Rffraction resulted in the title compound (10 mg, 33%).

¹H NMR (300 MHz, CD₃OD) δ 5.28 (d, J=5.4, 1), 4.73 (s, 1), 4.57–4.65 (m,1), 4.33–4.42 (m, 1), 4.05 (m, 1), 3.89 (s, 1), 3.53–3.57 (m, 2), 2.83(t, J=1.23, 1), 2.09 (s, 3), 1.63–1.84 (m, 5), 1.16–1.37 (m, 6), 0.93(m, 3). MS(ES+): 427 (M+1).

Example 56 Preparation of 4-Fluoro-4-propyl-pyrrolidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To a stirred solution of (2S, 4R)-4-hydroxyproline (Aldrich) (25 g, 108mmol) in methanol (50 mL) at 0° C. was added trimethylsilyldiazomethene(24.6 g, 216 mmol). The mixture was stirred at 0° C. for 1 hour. Theresidue obtained on removal of solvent and purification by columnchromatography using 50% ethyl acetate in hexanes (27 g, 100%) was usedin the next step. To oxalyl chloride (15 g, 118 mmol) in DCM (15 mL) at−78° C., DMSO (18.6 mL, 236 mmol) was added slowly over 15 minutes.After the completion of addition, the above product (2S,4R)-N-Boc-4-hydroxyproline methylester (26.5 g, 108 mmol) in DCM (100mL) was added at −78° C. for 20 minutes. Triethylamine (54.6 g, 540mmol) was added followed by stirring at room temperature for 2 hours.The reaction mixture was then washed with 10% aq HCl (200 mL) and theorganic layer was separated and dried over sodium sulfate. The crudeproduct obtained on removal of solvent was purified on silica gel columnchromatography using 50% EtoAc in hexanes to obtain4-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methylester (20 g, 78%).

¹H NMR (300 MHz, CDCl₃) δ 4.80 (m, 1), 3.88 (d, J=8.7, 2), 3.77 (s, 3),2.98 (m, 1), 2.58 (m, 1), 1.45 (s, 9); MS (ES+): 244 (M+1).

To a stirred solution of 4-oxo-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (1 g, 4.11 mmol) in THF (10 mL),tetraallyltin (1.08 mL, 4.52 mmol) in dry THF was added, then cooled to0° C. before borontrifluoride etherate (0.520 mL, 4.11 mmol) was addeddrop wise. The mixture was stirred at 0° C. for 1 h and then at roomtemperature for an additional 2 hours. Potassium fluoride (360 mg in 5mLwater) and celite (1 g) was added and the reaction mixture was stirredfor an hour. The reaction mixture was filtered and concentrated todryness and the residue was dissolved in DCM (200 mL), washed with water(100 mL) and brine 100 mL), dried over MgSO₄ and evaporated to dryness.The residue obtained on removal of solvent was purified by silica gelcolumn chromatography using 50% EtOAc in hexanes to obtain4-allyl-4-hydroxy-purrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (0.94 g, 80%).

¹H NMR (300 MHz, CDCl₃) δ 5.87 (m, 1), 5.19 (m, 2), 4.34 (m, 1), 3.75(d, J=4.8, 3), 3.50 (m, 3), 2.37 (m, 1), 2.21 (m, 1), 1.39 (d, J=12.9,9); MS (ES+): 308 (M+23).

To a stirred solution of DAST (1.06 g, 6.58 mmol) in DCM (10 mL) at −78°C., 4-allyl-4-hydroxy-purrolidine-1,2-dicarboxylic acid 1-tert-butylester 2-methyl ester (940 mg, 3.3 mmol) in dry DCM (10 mL) was addedslowly. The mixture was then stirred at −78° C. for 1 h, then at −10° C.for an additional 1 h. DCM (50 mL) was added, quenched with NH₄Cl (10%,150 mL) and the organic layer was separated, dried over sodium sulfateand evaporated to dryness. The residue obtained on removal of solventwas purified by silica gel column chromatography using 5% EtOAc inhexanes as eluent to obtain4-allyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (330 mg, 34%).

¹H NMR (300 MHz, CDCl₃) δ 5.82 (m, 1), 5.12 (m, 2), 4.43. (m, 1), 3.66(s, 3), 3.47 (m, 1), 2.37 (m, 1), 2.43 (m, 4), 1.37 (dd, J=4.5, 13.8,9); MS (ES+): 310 (M+23).

To a solution of 4-allyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (0.33 g, 1.15 mmol) in MeOH (15 mL)was added 10% Pd/C (40 mg) and hydrogenated at 1 atmosphere. Thecatalyst was removed by filtration through celite and washed withmethanol. To the product obtained on removal of solvent (330 mg, 1.15mmol) in THF (12 mL) was added lithium hydroxide monohydrate (60 mg,1.38 mmol). The reaction mixture was stirred at room temperatureovernight. THF was removed and the residue was taken up in ethyl acetate(50 mL), washed with 10% citric acid (100 mL) and brine (20 mL). Removalof solvent resulted in 4-allyl-4-propyl-pyrrolidine-1,2-dicarboxylicacid 1-tert-butyl ester 2-methyl ester (310 mg, 100%).

¹H NMR (300 MHz, CD₃OD) δ 4.43 (m, 1), 3.71 (m, 6), 2.51 (m, 2), 1.98(m, 3), 1.45 (m, 9), 0.96 (m, 3); MS (ES−): 274 (M−1).

To a solution of 4-allyl-4-propyl-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (310 mg, 1.15 mmol) in DMF (3 mL) at0° C., 7-Methyl MTL 2b (R¹=Me, R²=Me) (272 mg, 1.15 mmol), HBTU (469 mg,1.3 mmol) and DIEA (290 mg, 2.3 mmol) was added, left stirred at roomtemperature for 16 hours. DMF was removed and the residue obtained waspurified by 3% MeOH in DCM(40 mg, 93%). The product from the columnpurification was taken in DCE (6 mL), to which triethylsilane (0.16 mL),TFA (2 mL) and water (0.16 mL) was added and stirred at room temperaturefor 1.5 hours. Removal of solvent followed by purification on silica gelcolumn chromatography using 10% MeOH in DCM resulted in the titlecompound as isomeric mixtures with lower R_(f) fraction (160 mg, 50%).

¹H NMR (300 MHz, CD₃OD) δ 5.25 (d, J=5.7, 1), 4.46 (m, 1), 4.24 (dd,J=5.7, 10.2, 1), 4.08 (m, 2), 3.81 (d, J=2.4, 1), 3.52 (m, 3), 2.73 (m,1), 2.10 (m, 4), 1.88 (m, 2), 1.50 (m, 2), 0.99 (t, J=7.5, 3), 0.91 (dd,J=3.0, 6.9, 6); MS (ES+): 409 (M+1);) and higher Rf fraction (40 mg,12%). ¹H NMR (300 MHz, CD₃OD) δ 5.38 (d, J=5.4, 1), 4.46 (m, 1), 4.24(dd, J=2.7, 7.2, 1), 4.08 (m, 2), 3.81 (d, J=2.4, 1), 3.64 (m, 3), 2.73(m, 1), 2.11 (m, 4), 1.84 (m, 2), 1.47 (m, 2), 0.98 (t, J=7.5, 3), 0.91(dd, J=3.0, 6.9, 6); MS (METHOD ES+): 409 (M+1).

Example 57 Preparation of 4-Butyl-4-fluoro-pyrrolidine-2-carboxylic acid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

To ethyl acetylene (140 mg, 2.6 mmol) in THF (5 mL) at −78° C.,n-butyllithium (1.1 mL, 2.6 mmol) was added with stirring at −78° C. for1 hour. Then 4-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-methyl ester (prepared as described in the example 56) (570 mg, 2.3mmol) in THF (5 mL) was added at −78° C. with stirring for 2 h, thereaction mixture was then allowed to warm to −40° C. over 1 hour. Thereaction mixture was extracted with EtOAc (20 mL), washed with saturatedNH₄Cl (5 mL) and dried over sodium sulfate. Purification of the crudeproduct was carried out by silica gel chromatography using 50% EtOAc inhexane to obtain the 4-butyl4-hydroxy-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (520 mg, 73%). To the DAST (556 mg,3.4 mmol) in DCM (5 mL) at −78° C., was added a solution of the aboveester (520 mg, 1.7 mmol) in DCM (5 mL) at −78° C. and stirred for 1hour. The reaction mixture was extracted with DCM (50 mL) and washedwith NaHCO₃ (30 mL, 10%). The product obtained after removal of solventwas purified by silica gel chromatography using 5% EtOAc in hexanes toobtain 4-butyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butylester 2-methyl ester (276 mg, 52%).

¹H NMR (300 MHz, CD₃OD) δ 4.41 (m, 1), 3.83 (m, 1), 3.71 (s, 3), 3.45(m, 1), 2.55–1.54 (m, 8), 1.39 (m, 9), 0.89 (m, 3); MS (ES+): 326(M+23).

To a solution of 4-butyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester 2-methyl ester (270 mg, 0.89 mmol) in THF (12 mL) andwater (4 mL), was added lithium hydroxide monohydrate (45 mg, 1.07mmol). The reaction mixture was stirred at room temperature for 16hours. THF was removed under vacuum and the residue was taken up inethyl acetate (150 mL), washed with 10% citric acid (100 mL) and brine(20 mL). Removal of solvent provided4-butyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester(260 mg, 100%).

¹H NMR (300 MHz, CD₃OD) δ 4.32 (m, 1), 3.72 (m, 2), 2.58 (m, 2),2.10–1.63 (m, 6), 1.42 (m, 9), 0.93 (t, J=6.6, 3); MS (ES−): 288 (M−1).

To a solution of 4-butyl-4-fluoro-pyrrolidine-1,2-dicarboxylic acid1-tert-butyl ester (135 mg, 0.46 mmol) in DMF (3 mL) at 0° C., 7-MethylMTL 2b (R¹=Me, R²=Me) (135 mg, 0.46 mmol), HBTU (194 mg, 0.51 mmol),DIEA (120 mg, 0.93 mmol) was added, left it at room temperature for 16hours. The product obtained after removing DMF and purification bycolumn chromatography using 5% MeOH in DCM (189 mg, 77%) was taken inDCE (6 mL). Triethylsilane (0.16 mL), TFA (2 mL) and water (0.16 mL) wasadded, stirred at room temperature for 1.5 hours. The residue obtainedon removal of solvent was purified by column chromatography using 10%MeOH in DCM to obtain the title compound (156 mg, 96%).

¹H NMR (300 MHz, CD₃OD) δ 5.26 (d, J=5.7, 1), 4.55 (m, 1), 4.27 (dd,J=3.3, 10.2, 1), 4.08 (m, 2), 3.82 (d, J=3.0, 1), 3.58 (m, 3), 2.79 (m,1), 2.22 (m, 1), 2.10 (s, 3), 1.89 (m, 3), 1.40 (m, 4), 0.91 (m, 9); MS(ES+): 423 (M+1).

Example 58 4-Fluoro-4-propyl-pyrrolidine-2-carboxylic acid[2-hydroxy-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

N-(tert-butoxycarbonyl)-4-fluoro-4-propyl-L-proline was prepared asdescribed in the previous example (except using n-propyl lithium in theplace of n-butyl lithium) (164 mg, 0.57 mmol) was suspended in dryacetonitrile (4 mL). Triethylamine (332 μL, 3.02 mmol) was added and thereaction mixture was cooled to 0° C. Isobutyl chloroformate (78 μL, 0.57mmol) was added and after 10 min the reaction was allowed to warm to 4°C. After 1.5 h a solution of MTL (151 mg, 0.57 mmol) in 1:1 acetone:water (4 mL) was added and the reaction mixture was stirred for 10 h atrt. The reaction mixture was evaporated to dryness and chromatographedon silica 95:5 dichloromethane/MeOH to 95:8 dichloromethane/MeOH toprovide the product as a colorless oil (137 mg, 45%): TLC Rf 0.32 (9:1dichloromethane/MeOH).

To a solution of the above boc protected lincosamide (125 mg,) in DCM(2.0 mL) was added a solution of DCE (10.0 mL), trifluoroacetic acid (5mL) methyl sulfide (0.3 mL), and water (0.3 mL). The reaction mixturewas stirred at rt for 40 min then diluted with DCE (25.0 mL). Thesolvent was removed under vacuum and co-evaporated with DCE twice. Theresidue was purified by chromatography on fluorosil 20% MeOH (0.25M NH₃)in DCM to provide the product as a colorless solid (30.0 mg, 30%).

Example 59 Preparation of 4-(2-methoxyethoxy)-piperidine-2-carboxylicacid[2-methyl-1-(3,4,5-trihydroxy-6-methylsulfanyl-tetrahydro-pyran-2-yl)-propyl]-amide

The title compound was made using the synthetic sequence found ingeneral Method S starting from 4-hydroxypyridine-2-carboxylic acid,substituting 1-bromo-2-methoxy-ethane as the alkylating agent.

Compound 15a (R¹⁰=2-methoxyethyl): ¹H NMR (300 MHz, CD₃OD) δ 8.40 (d,J=6.0, 1), 7.69 (d, J=2.4, 1), 7.20 (dd, J=2.7, 6.3, 1), 4.35 (m, 2),3.80 (m, 2), 3.40 (s, 3). MS (ES−): 196(M−1).

Compound 15b (R¹=Me, R²=Me, R¹⁰=2-methoxyethyl): ¹H NMR (300 MHz, CD₃OD)δ 8.43 (d, J=5.7, 1), 7.65 (d, J=2.4, 1), 7.12 (dd, J=2.4, 5.7, 1), 5.27(d, J=5.4, 1), 4.10–4.87 (m, 4), 3.85 (d, J=3.3, 1), 3.77 (m, 2), 3.55(m, 1), 3.41 (s, 3), 2.26 (m, 1), 2.11 (s, 1), 0.998 (m, 6). MS (ES⁺):431 (M+1).

Title compound (10 mg, 10%). ¹H NMR (300 MHz, D₂O) δ 5.18 (d, J=6.0, 1),4.00 (m, 3), 3.70 (m, 1), 3.56 (m, 1), 3.45 (m, 3), 3.26 (m, 1), 3.16(m, 3), 3.10 (m, 1), 2.80 (m, 1), 2.48 (m, 1), 2.22 (m, 1), 1.96 (m, 4),1.17–1.72 (m, 4), 0.70 (m, 6); MS (ES⁺): 437 (M+1).

Examples 60 to 65, as indicated in Table 1 above, were prepared usingmethods and techniques describe herein utilizing commercially availablestarting materials where appropriate.

The following Examples may be used to test compounds of this invention.

Example A Susceptibility Testing

Compounds were tested following the microdilution method of NCCLS(National Committee for Clinical Laboratory Standards. Methods fordilution antimicrobial susceptibility tests for bacteria that growaerobically; Approved standard—fifth edition. NCCLS document M7-A5,NCCLS, Wayne, Pa. 2000; National Committee for Clinical LaboratoryStandards. Methods for antimicrobial susceptibility testing of anaerobicbacteria; Approved standard—fifth edition. NCCLS document M11-A4, NCCLS,Wayne, Pa. 2001). Assays were performed in sterile plastic 96-wellmicrotiter trays with round bottom wells (Greiner).

Compound Preparation

Stock solutions of test compounds and control antibiotics are preparedat 10 mg/mL in DMSO. Serial 2-fold dilutions of each drug are performedin a microtiter plate across each row using DMSO as solvent at 100-foldthe desired final concentration. Wells in columns #1–11 contain drug andcolumn #12 was kept as a growth control for the organism with no drug.Each well in the mother plate is diluted with sterile deionized water,mixed, and volumes of 10 μL distributed to each well in the resultingassay plates.

Preparation of Inoculum

Stock cultures were prepared using the Microbank™ method (Pro-LabDiagnostics) and stored at −80° C. To propagate aerobic strains, onebead was removed from the frozen vial and aseptically streaked ontoTrypticase Soy Agar (Difco), Chocolate Agar (Remel) or Blood Agar(Remel) which were incubated at 35° C. overnight. Anaerobes werecultivated in Brucella Agar (Remel) supplemented with hemin and vitaminK and incubated in anaerobiosis using an Anaerobic Jar (Mitsubishi) at35° C. for 24 to 48 h. Standardized inocula were prepared using thedirect colony suspension method according to NCCLS guidelines (NationalCommittee for Clinical Laboratory Standards. Methods for dilutionantimicrobial susceptibility tests for bacteria that grow aerobically;Approved standard—fifth edition. NCCLS document M7-A5, NCCLS, Wayne, Pa.2000; National Committee for Clinical Laboratory Standards. Methods forantimicrobial susceptibility testing of anaerobic bacteria; Approvedstandard—fifth edition. NCCLS document M11-A4, NCCLS, Wayne, Pa. 2001).Isolated colonies were selected from an 18–24 h agar plate andresuspended in 0.9% sterile saline to match a 0.5 McFarland turbiditystandard. The suspension was used within 15 min. of preparation.

Streptococcus pneumoniae VSPN1001 Streptococcus pneumoniae ATCC 49619Streptococcus pneumoniae VSPN3026 Streptococcus pneumoniae R6xStreptococcus pneumoniae VSPN4054 Streptococcus pneumoniae 488 KStreptococcus pneumoniae VSPN4021 Streptococcus pneumoniae 9Staphylococcus aureus VSAU1017 Staphylococcus aureus SmithStaphylococcus aureus VSAU1003 Staphylococcus aureus ATCC 25923Staphylococcus aureus VSAU4020 Staphylococcus aureus 125 Staphylococcusaureus VSAU4048 Staphylococcus aureus 85-EPI Staphylococcus aureusVSAU4065 Staphylococcus aureus VSAU4065 Staphylococcus epidermidisVSEP1001 Staphylococcus epidermidis ATCC 12228 Enterococcus faecalisVEFL1003 Enterococcus faecalis ATCC 51299 Enterococcus faecium VEFA1005Enterococcus faecium BM4147.1 Haemophilus infuenzae VHIN1003 Haemophilusinfuenzae ATCC 49766 Haemophilus infuenzae VHIN1004 Haemophilusinfuenzae ATCC 31517 Haemophilus infuenzae VHIN1005 acr Haemophilusinfuenzae LS-2 Moraxella catarrhalis VMCA1001 Moraxella catarrhalis ATCC25238 Escherichia coli VECO2096 Escherichia coli MG1655 Escherichia coliVECO2526 to1C Escherichia coli MG1655 to1C Bacteroides fragilis VBFR1001Bacteroides fragilis ATCC 25285 Bacteroides thetaiotaomicron VBTH 1001Bacteroides thetaiotaomicron ATCC #29741 Clostridium difficile VCDI1001Clostridium difficile ATCC 9689Preparation of Assay Plates for MICs Preparation of Assay Plates forMICs

Media were prepared at 1.1× concentration. Mueller-Hinton Broth MHB(Difco) supplemented with Ca++ and Mg++ as recommended by NCCLS, MHBsupplemented with 5% horse lysed blood, HTM Broth (Remel), or Brucellabroth (Remel) supplemented with hemin and vitamin K. For each organism,the standardized suspension was diluted into appropriate growth mediumin a sterile reservoir. After mixing, wells in the drug-containing assayplates were inoculated with a volume of 90 μl. Thus, for each MICdetermination, each well contains a final volume of 100 μL with aninoculum size of approximately 5 * 105 cfu/mL and no more than 1% DMSO.

Interpretation of MIC

The completed microtiter plates were incubated 16–20 h at 35° C. inambient air for aerobes, and at 35° C. for 46–48 h or in an anaerobe jar(Mitsubishi) for anaerobes. Optical density of each well was determinedat 600 nm using a VersaMax Microplate reader (Molecular Devices,Sunnyvale, Calif.). The MIC was defined as the lowest drug concentrationcausing complete suppression of visible bacterial growth.

Example B Efficacy in Murine S. aureus Septicemia

Efficacy studies were performed in an S. aureus murine septicemia modelaccording to models published elsewhere (Goldstein, B. P., G. Candiani,T. M. Arain, G. Romano, I. Ciciliato, M. Berti, M. Abbondi, R. Scotti,M. Mainini, F. Ripamonti, and et al. 1995. Antimicrobial activity of MDL63,246, a new semisynthetic glycopeptide antibiotic Antimicrob AgentsChemother. 39:1580–1588.; Misiek, M., T. A. Pursiano, F. Leitner, and K.E. Price 1973. Microbiological properties of a new cephalosporin, BL-S339:7-(phenylacetimidoyl-aminoacetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)ceph-3-em-4-carboxylic acid Antimicrob Agents Chemother.3:40–48).

Compound Preparation

Compounds were dissolved in 2% Tween 80 for oral dosing or 0.9% NaClsolution for intravenous dosing. Compounds were administered at 1 hourafter bacterial inoculation. Vancomycin or ampicillin were used ascontrols.

Efficacy Model

Male or female ICR mice weighing 22±2 g from MDS Pharma Services wereused for the evaluation. Food and water was given ad libitum. Groups of6 mice weighing 22±g were used for the experiment. Mice were inoculatedintraperitoneally with Staphylococcus aureus Smith at 4 104 CFU in 0.5mL of Brain Heart Infusion Broth (Difco) containing 5% mucin (Sigma).Mortality was recorded once daily for 7 days following bacterialinoculation.

While the invention has been described and illustrated herein byreferences to various specific material, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

Example C In Vivo Animal Model

In vivo activity of various compounds of the subject invention wasevaluated in a standard Staphylococcus aureus septicemia model (MDSPharma Services, Bothell, Wash.).

Male ICR-derived mice (ICR is a strain of out-bred mice) provided by MDSPharma Services animal breeding center were inoculated intraperitonealywith LD₉₀₋₁₀₀ of Staphylococcus aureus. (Smith; ATCC19636) in 0.5 mL BHIbroth containing 5% mucin (Sigma). Compounds were formulated in 2% Tween80 (Sigma) and single doses were administered orally one hour afterbacterial inoculation. Mortality was monitored daily for seven days.

In previous studies, the oral ED₅₀ (i.e., concentration that protected50% of the mice) was determined to be 19.9 mg/kg for clindamycin, acommercially available lincosamide (Sigma). To screen the compounds ofthis invention that were tested, compounds were administered at 10 mg/kgto a group of eight ICR mice and the number of survivors at thatconcentration was compared to clindamycin. Results are presented in thetable below.

Compound Tested No. of (Indicated by surviving mice Example No.) at 10mg/kg Clindamycin 6 17 1 34 1 35 2 35 0

It is assumed that when compound from example 35 is tested at a higherdose, the number of surviving mice would increase.

1. A compound of the formula:

wherein R² and R³ are independently selected from the group consistingof H, alkyl, fluoro; and R⁶ is selected from the group consisting of H,hydroalkyl, —C(O)O-alkylene-cycloalkyl, —C(O)O-alkyl, and —C(O)O-aryl;or prodrugs, tautomers or pharmaceutically acceptable salts thereof. 2.The compound of claim 1, wherein R² is H, R³ is CH₃, and R⁶ is H.
 3. Thecompound of claim 1, wherein R² is H and R³ is CH₃.
 4. The compound ofclaim 1, wherein R² is H.
 5. The compound of claim 1, wherein R⁶ is2-hydroethyl.
 6. The compound of claim 1, wherein R⁶ isfluorenyl-methyleneoxy carbonyl.
 7. The compound of claim 1, wherein R⁶is ethoxycarbonyl.
 8. The compound of claim 1, wherein R⁶ isphenoxycarbonyl.
 9. The compound of claim 1, wherein R² and R³ are F.10. A compound of the formula:

wherein R⁹ is selected from ethyl, butyl, isobutyl, pentyl,3-fluoropropyl, 3-difluoropropyl, 3-difluorobutyl, 4-fluorobutyl,4-difluoropentyl, 5-difluoropentyl, 5-fluoropentyl, butoxy, pentoxy,2-fluoroethoxy, 3-fluoropropoxy, 3-trifluoropropoxy, or 4-fluorobutoxy;or prodrugs, tautomers or pharmaceutically acceptable salts thereof. 11.The compound of claim 10, wherein R⁹ is ethyl.
 12. The compound of claim10, wherein R⁹ is butyl.
 13. The compound of claim 10, wherein R⁹ isisobutyl.
 14. The compound of claim 10, wherein R⁹ is pentyl.
 15. Thecompound of claim 10, wherein R⁹ is 3-fluoropropyl.
 16. The compound ofclaim 10, wherein R⁹ is 3-difluoropropyl.
 17. The compound of claim 10,wherein R⁹ is 3-difluorobutyl.
 18. The compound of claim 10, wherein R⁹is 4-fluorobutyl.
 19. The compound of claim 10, wherein R⁹ is4-difluoropentyl.
 20. The compound of claim 10, wherein R⁹ is5-difluoropentyl.
 21. The compound of claim 10, wherein R⁹ is5-fluoropentyl.
 22. The compound of claim 10, wherein R⁹ is butoxy. 23.The compound of claim 10, wherein R⁹ is pentoxy.
 24. The compound ofclaim 10, wherein R⁹ is 2-fluoroethoxy.
 25. The compound of claim 10,wherein R⁹ is 3-fluoropropoxy.
 26. The compound of claim 10, wherein R⁹is 3-trifluoropropoxy.
 27. The compound of claim 10, wherein R⁹ is4-fluorobutoxy.
 28. The compound of claim 1, having the formula:

wherein R², R³, and R⁶ are as defined in claim
 1. 29. The compound ofclaim 10 having the formula:

wherein R⁹ is as defined in claim 10.